design and development of protein-polymer ... - unibas.ch thesis pascal tanner 2013.pdf ·...

i

Design and development of protein-polymer

assemblies to engineer artificial organelles

Inauguraldissertation

zur Erlangung der Würde eines Doktors der Philosophie

vorgelegt der

Philosophisch-Naturwissenschaftlichen Fakultät

der Universität Basel

von

Pascal Tanner

aus Werthenstein, Luzern

Basel, 2013

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Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät

auf Antrag von

Prof. Dr. Cornelia Palivan (Universität Basel),

Prof. Dr. Wolfgang Meier (Universität Basel)

und

Prof. Dr. Marcus Textor (ETH Zürich)

Basel, den 17.09.2013

Prof. Dr. Jörg Schibler

(Dekan)

Engineering artificial organelles

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Abstract

Molecular engineering by means of the development of artificial

organelles that can be implanted in cells to treat pathological conditions

or to support the design of artificial cells has been regarded as a major

goal in medical research. In order to achieve this goal, active compounds,

such as enzymes, proteins and enzyme-mimics were encapsulated or

entrapped in polymer compartments to create highly active hybrid

nanoreactors. However, probably due to the early stage of research, only

few of these nanoreactors were active in cells, and none was proven to

mimic a specific natural organelle. In the present thesis, the engineering

of polymeric vesicles for the development of artificial organelles

mimicking natural peroxisomes is accomplished. In addition, a detailed

study of the permeabilisation of polymeric vesicle membranes by

incorporation of ion-channels is elaborated. The detailed study of metal-

functionalised polymeric vesicle membranes for targeting approaches –

an important factor for site specific action of e. g. artificial organelles –

gives a further key aspect. These three parts of fundamental research

play a key role in further development of advanced nanoreactors to be

used as specific artificial organelles. In the first study, we designed

systems that contained two different highly active antioxidant enzymes,

which worked in tandem in polymer nanovesicles. The membrane of the

nanovesicles was specifically permeabilised by the incorporation of

natural channel proteins and the functional nanoreactor was optimised

with regard to properties and function of natural peroxisomes. These non-

toxic artificial peroxisomes combat oxidative stress in cells after cell-

uptake, which prolong cell life-time and which can be regarded as the first

instance of treatment of various pathologies (e.g. arthritis, Parkinson’s,

cancer, AIDS) effectively controlled by conditions of oxidative stress. The

second study proofs the successful incorporation of small ion-channels in

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the vesicle membrane. These ion-channels are specifically permeable for

monovalent cations, for example to design a pH-sensitive artificial

organelle, where the active compound can be switched on/off

respectively, depending on the conditions of the environment. The third

project focussed on the decoration of metal-functionalised polymersomes

with a small model peptide, a His6-tag, in order to characterise the change

of the metal coordination site upon binding of the model peptide. This

provided important information about the binding behaviour of ligands in

processes of molecular recognition, a key principle in nature.

Understanding of specific and efficient binding will allow to further target

the polymersomes to specific locations to either release their content,

such as drugs, or to act as an artificial organelle.


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Table of content

1. INTRODUCTION ..................................................................................................... 1 1.1. CONCEPT OF MOLECULAR ENGINEERING ..................................................................... 3 1.2. BUILDING BLOCKS FOR MOLECULAR ENGINEERING .................................................... 4

1.2.1 Bio-related properties of the amphiphilic di- and tri-block copolymers used in this thesis .......................................................................................... 6

1.3. AMPHIPHILIC BLOCK COPOLYMER SUPERSTRUCTURES ............................................ 7 1.3.1. Principle of self-assembly ..................................................................................... 9 1.3.2. Polymeric vesicles (polymersomes) .............................................................. 10 1.3.3. Polymersome preparation methods ............................................................. 11 1.3.4. Characterisation methods of polymersomes ............................................ 12

1.4. CONCEPT OF NANOREACTORS BASED ON AMPHIPHILIC BLOCK COPOLYMERS ... 14 1.4.1. Requirements for nanoreactors used in medical applications ......... 16 1.4.2. Active compounds supporting nanoreactor functionality ................. 18 1.4.3. Incorporation of membrane proteins and ion-channels in amphiphilic block copolymer polymersome membranes ................................. 19 1.4.4. Engineering artificial organelles as cell implants based on nanoreactors ......................................................................................................................... 21

1.5. CONCEPT OF NANOCARRIERS FOR DRUG/PROTEIN DELIVERY BASED ON

AMPHIPHILIC BLOCK COPOLYMERS ..................................................................................... 22 1.6. RELEVANCE OF REACTIVE OXYGEN SPECIES (ROS) IN OXIDATIVE STRESS ........ 23

1.6.1. Natural defence mechanism ............................................................................. 25 1.6.2. Antioxidant therapy ............................................................................................. 27

1.7. CONCEPT OF MOLECULAR RECOGNITION .................................................................. 28 1.7.1. Molecular recognition strategies in nature .............................................. 29 1.7.2. Targeting strategies ............................................................................................. 30 1.7.3. Metal-chelation: NTA and trisNTA ................................................................ 30 1.7.3. His-tags and His-tagged molecules ............................................................... 32

2. COMBAT SUPEROXIDE ANION RADICALS BY ANTIOXIDANT NANOREACTORS ....................................................................................................... 35

2.1. MOTIVATION AND CONCEPT ....................................................................................... 36 2.2. DESIGN OF AN ENZYMATIC CASCADE REACTION TO FIGHT OXIDATIVE STRESS .. 38 2.3. DESIGN AND CHARACTERISATION OF ANTIOXIDANT NANOREACTORS ................ 39

2.3.1. Optimised encapsulation of individual enzymes in polymersomes 41 2.3.2. Activity assay of individual enzyme species in solution and in polymersomes ....................................................................................................................... 48 2.3.3. Optimised co-encapsulation of SOD and LPO in polymersomes ...... 52 2.3.4. Multi-enzyme kinetics in a solution that simulates polymersome conditions ............................................................................................................................... 55

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2.3.5. Multi-enzyme kinetics in situ inside polymersomes .............................. 56 2.4. COMBATING SUPEROXIDE ANION RADICALS IN CELLS BY ARTIFICIAL

PEROXISOMES ......................................................................................................................... 58 2.4.1. Integration of artificial peroxisomes in cells............................................ 60

2.5. CONCLUSION .................................................................................................................. 69

3. DESIGN OF PH-TRIGGERED NANOREACTORS ........................................... 71 3.1. MOTIVATION AND CONCEPT........................................................................................ 72 3.2. EVIDENCE OF FUNCTIONAL ION-CHANNEL INCORPORATION IN LIPOSOMES ....... 72 3.3. EVIDENCE OF FUNCTIONAL ION-CHANNEL INCORPORATION IN POLYMERSOMES

.................................................................................................................................................. 74 3.3.1. Ion-channel formation (H+ kinetics) as a function of gramicidin concentration in polymersome membranes .......................................................... 78

3.4. PH-TRIGGERED NANOREACTORS ................................................................................ 79 3.5. CONCLUSION .................................................................................................................. 82

4. METAL-FUNCTIONALISED POLYMERSOMES FOR MOLECULAR RECOGNITION ............................................................................................................ 83

4.1. MOTIVATION AND CONCEPT........................................................................................ 83 4.2. FORMATION OF CU(II)-TRISNTA FUNCTIONALISED POLYMERSOMES ................ 86 4.3. HIS6-TAGS BINDING TO CUII-TRISNTA POLYMERSOMES ....................................... 88 4.4. STRUCTURE OF THE COORDINATION SPHERE OF CUII IN HIS6-TAG-CUII-TRISNTA

POLYMERSOMES IN COMPARISON TO MODEL SYSTEMS .................................................... 94 4.5. CONCLUSION ............................................................................................................... 106

5. OVERALL CONCLUSION .................................................................................. 109

6. EXPERIMENTAL SECTION .............................................................................. 111

7. APPENDIX ........................................................................................................... 129

8. REFERENCES ...................................................................................................... 135

9. ACKNOWLEDGEMENTS .................................................................................. 151

10. ABBREVIATIONS ............................................................................................ 153

11. CURRICULUM VITAE ..................................................................................... 157


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1. Introduction

The design of new, functional and smart materials or assemblies is

mainly driven to understand biological systems in more detail and to

mimic biological structures and functions. There is a high need to obtain a

profound knowledge of the working principle of biological systems in order

to repair many diseases on molecular basis. Furthermore, the

understanding of the complexity, specificity and accuracy of biologic

pathways forms the basis of technological progress. Hence, it is a key

challenge in life- and nanosciences to fuse biomolecules, such as

enzymes or proteins, with synthetic materials, for example block

copolymers, in order to create new, complex bio-synthetic nanodevices.

The combination of the specificity and efficiency of biological molecules

with the robustness and the possibility of tailoring synthetic materials

allows the design of efficient systems in terms of activity, sensitivity and

responsiveness.1,2 In addition, this strategy allows protecting sensitive

compounds, for example enzymes, proteins, or nucleic acids, while

providing optimal working conditions − the same principle as in the

compartmentalisation strategy in nature. Successful protection was put

into practice in recent years by shielding various biological compounds in

nanometre-sized supramolecular assemblies. The purpose of shielding

was already commercialized by using liposomes, which are bio-adapted,

lipid-based supramolecular assemblies. However, limitations in the use of

liposomes such as increased, intrinsic mechanical instability and low

blood circulation life-time could only be partially solved through their

combination with polymers such as polyethylene glycol (PEG), which has

shown to have “stealth” behaviour (by means of no association with non-

targeted serum and tissue proteins thus being able to evade the immune

system).3

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Consequentially, polymer supramolecular structures in the form of

particles, micelles, vesicles and films are of particular interest, because

they can adapt their properties to support combination with biological

molecules while preserving material properties such as stability and

mechanical robustness.4,5 Polymer supramolecular assemblies exhibit

morphologies and properties, which can be adapted by choosing from a

variety of highly diverse block copolymers. By triggering the block length

of each block unit of the block copolymer, the entire molecular

architecture is provided. In addition, properties such as charge, flexibility

or stimuli-responsiveness can be tuned for further system integration.6 In

conclusion, this outstanding adaptability makes these assemblies very

versatile for a wider range of applications.7,8

Elegant combinations of nanometre-sized, supramolecular assemblies

that protect active compounds and support their in situ activity are

polymer nanoreactors.9,10 Nanoreactors based on polymer vesicles

encapsulating active molecules and with a membrane rendered

permeable for small molecules provide fully active compounds in

biologically relevant conditions inside the carrier over long time. This

approach eliminates the drawbacks related to conventional drug carriers,

where active compounds may act only after their release from the carrier

or are released into a different biological compartment, other than

desired.11 When nanoreactors are used in vitro, sensitivity and overall

activity are essential properties to be coped with, while for in vivo

applications (therapeutics or theranostics) more complex requirements

are imposed in terms of nanoreactor-assemblies properties, in situ

functionality, and biodistribution.

Our aim here was to engineer artificial organelles focussing on three

aspects: i.) design and development of a functional cascade reaction


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inside polymersomes ii.) engineering triggerable nanoreactors and iii.)

studying a specific molecular recognition process in detail.

i.) In a bio-inspired approach, the nanoreactor design was used to

mimic a specific artificial organelle by the combination of two different

antioxidant enzymes, superoxide dismutase together with lactoperoxidase

or catalase. Implementation of the cascade reaction into the

polymersomes enabled the use of the SOD/LPO co-encapsulated as

artificial peroxisomes inside cells to repair the condition of oxidative

stress.

ii.) Another approach of nanoreactor design, focussing on pH-triggered

nanoreactors, was realised by incorporation of an ion-channel, gramicidin,

in the membrane of pH-sensitive acid phosphatase-encapsulated

polymersomes. Feasibility of pH-stimulation of the enzymes in vesicles

was proven.

iii.) We used molecular recognition interactions and metal-

functionalised block copolymers in order to form vesicles with specific

metal points at the outer vesicle surface. Binding studies of small ligands

and detailed investigation of the metal-centre coordination spheres before

and after binding gave information about optimal binding conditions.

1.1. Concept of molecular engineering

Controlling the structure of materials/compounds at molecular

dimensions opens the possibility to produce nanosystems or

nanofactories, which do not occur in nature having for instance new

functionalities or are mimics of biological systems. In general, two

important strategies in molecular engineering can be followed as bottom

up approaches in the design of nanofactories. One involves highly

developed technologies such as a scanning tunnelling microscopy, which

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enables the control of individual molecules and allows arranging them in a

functional way in order to create an active nanosystem. The other

strategy makes use of the self-assembly principle naturally evolved in

biological systems to build more complex systems. The self-assembly

principle has the advantage that molecules assemble into structures

based on molecular interactions such as hydrogen bonds, Van der Waals

or electrostatic interactions without the need of expensive techniques and

time-consuming procedures. Complex templates can be engineered by

using molecules, for example, lipids or amphiphilic block copolymers. In

order to design functional nanofactories, active molecules such as

enzymes, proteins or enzyme-mimics need to be combined with the self-

assembled structure. Preservation of the functionality of the active

molecules during this process is a key step to engineer functional

nanodevices such as nanoreactors. This requires the working principle

and optimised conditions of the active molecule to be studied in detail.

After proof of concept, application of the engineered nanofactories either

in the technological or biomedical domain has to fulfil further

requirements, addressing for example maximising efficiency in sensor

systems or increased efficacy and low immunotoxicity in therapeutic

applications.

1.2. Building blocks for molecular engineering

Different building blocks to engineer supramolecular assemblies as

template structures for medical applications can be used: Biopolymers,

synthetic polymers or combinations of bio- and synthetic polymers.

Biopolymers include lipids, polysaccharides, polypeptides and

polynucleotides. Their inherent biocompatibility and biodegradability in


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human bodies and cheap production qualify them for technological

applications such as carrier systems.12

The category of synthetic polymers can be divided into homopolymers,

polyelectrolytes and block copolymers.10,13-15 Homopolymers are built

from covalently attached identical repeating units in linear or branched

form. Polyelectrolytes have repeating polymer units with charged

functional groups.

Fig. 1 Amphiphilic block copolymer architectures: A) AB-diblock; B) ABA-

triblock; C) ABC-triblock; D) dendrimer; E) star-shape; F) linear grafted shape.

This thesis is mainly based on amphiphilic block copolymers. Block

copolymers belong to a special class of synthetic macromolecules

consisting of at least two different groups of repeating structural units

called homopolymers. The different homopolymer units are either

covalently linked or are linked by a junction block, which is an

intermediate and non-repeating spacer.16 Amphiphilic block copolymers

are composed of at least two subunits, where one homopolymer subunit

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has hydrophilic (water soluble) properties while the other has hydrophobic

(water insoluble) character. The homopolymer block is abbreviated for

clarity as A, B, C etc., and their order in the block copolymer is directly

used for its classification. According to this classification, block copolymer

architectures such as AB – diblock, ABA – triblock, ABC – triblock,

dendrimer shape, star-shape and grafted are shown in Fig. 1. These

architectures can have either linear, cyclic, multi-arm star, comb, hyper-

branched or dendritic shapes.17

1.2.1 Bio-related properties of the amphiphilic di- and tri-block copolymers used

in this thesis

In this work, three different kinds of amphiphilic block copolymer were

employed: A triblock copolymer consisting of an amphiphilic poly(2-

methyloxazoline)-b-poly-(dimethylsiloxane)-b-poly(2-methyloxazoline)

(PMOXA-PDMS-PMOXA), a diblock copolymer based on poly(2-

methyloxazoline)-b-poly-(dimethylsiloxane) (PMOXA-PDMS) and a

diblock copolymer made of poly(butadiene)-b-poly(ethylene oxide) (PB-b-

PEO). The biologically relevant properties of the individual

homopolymers are summarised here:

PMOXA: PMOXA is often used as the hydrophilic block in either AB

diblock or ABA triblock copolymers as building block in supramolecular

assemblies for biological applications.2,11 Recent studies on the

biodistribution and excretion of well-defined radiolabelled PMOXA in mice

demonstrated no accumulation in tissue and rapid clearance from the

bloodstream, thus apportion a good biocompatibility of biodegradability of

PMOXA.18 Cell-viability measurements of block copolymers containing

PMOXA indicated low toxicity, which makes it an ideal candidate to build

functional artificial organelles.


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PDMS: Silicones, and in this special case, PDMS are widely used in

medical applications, such as breast implants.19 As it is non-irritating and

non-sensitising, it shows good biocompatibility. In block copolymers, it is

widely used as the hydrophobic block. Also block copolymers containing

PDMS were widely used to build supramolecular assemblies such as

nanovesicles for diagnostic or therapeutic applications.

PEO: PEO is widely applied in drug delivery applications. It has a good

biocompatibility, hydrophilicity, versatility and stealth behaviour.18 Studies

have demonstrated a protein repellent character.20 This linear polymer

can be easily modified for example by covalent binding of a metal-

chelating agent for targeting purpose.21

PB: PB has vinyl groups attached to the backbone and is soluble only

in organic solvents such as acetone, but insoluble in water.22 PB is often

used as the hydrophobic block in amphiphilic block copolymers.21-23 The

monomer 1, 3-butadiene is highly toxic. It is expected to be involved in

cardiovascular disease, leukaemia and other cancer as described by the

national pollutant inventory. Therefore its use in bio-related applications is

not recommended. Because PB was used in this thesis as a part of the

diblock copolymer PB-b-PEO, which formed nanovesicles, in order to

study the geometry changes of the metal-functionalised part in the

process of molecular recognition, the potential toxicity of this block

copolymer was no matter of interest.

1.3. Amphiphilic block copolymer superstructures

Amphiphilic molecules, such as lipids, surfactants or amphiphilic block

copolymers, possess the ability to form self-assembled superstructures in

aqueous media. The hydrophilic part and the hydrophobic part thereby

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Fig. 2 Self-assembled structures based on block copolymers and surfactants:

spherical micelles, cylindrical micelles, vesicles, fcc- and bcc-packed spheres (FCC,

BCC), hexagonally packed cylinders (HEX), various minimal surfaces (gyroid, F

surface, P surface), simple lamellae (LAM), as well as modulated and perforated

lamellae (MLAM, PLAM). Reprinted with permission from Ref. 24 Copyright 2002

WILEY-VCH Verlag GmbH & Co. KGaA.

describe the overall amphilicity of the molecule. The two blocks were

covalently linked in order to avoid macrophase separation,24 which

occurs for example by water and oil emulsions. At defined conditions,

molecular ordered superstructures were formed via self-assembly

depending on the insoluble to soluble ratio, the molecular weight and the

concentration.25,26 In this case, different morphologies such as micelles,

vesicles, tubes, lamellar structures, etc. are obtained (Fig. 2). The type of

morphology can be also modulated by different solvents, where only one

block of the amphiphile is soluble.27

Amphiphilic block copolymer superstructures are well suited to mimic

biological structures in order to engineer new functional materials for


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therapeutic, diagnostic and theranostic applications due to serious

advantages compared to their natural counterparts (e.g. lipids).

1.3.1. Principle of self-assembly

The self-assembly process is a fundamental principle in nature to

organise molecules from a disordered system to highly organised

structures or patterns without the aid of external trigger factors. Well-

ordered biological assemblies can be formed spontaneously by the self-

assembly process. For example lipid bilayers, the matrix of cell

membranes, are formed by the self-assembly of lipids. Other self-

assembled structures are proteins, nucleic acids and viruses. The self-

assembly process is essential for these structures to fulfil their biological

function.

The self-assembly process is based on intermolecular and/or

intramolecular interactions, whereas the process is mainly driven by non-

covalent hydrophobic interactions. During the self-assembly process in

aqueous solution, the hydrophobic parts of amphiphiles preferentially

align with each other, which is favourable compared to the interaction with

water leading to an increased entropy (hydrophobic effect).28 In order to

predict the self-assembly behaviour of amphiphilic block copolymers

containing hydrocarbons, the packing parameter P describes the shape of

the molecules (equation 1).29

⁄ (1)

V is described as the hydrophobic chain volume, l as the preferred

length and a0 the area, which is occupied by the hydrophilic head group of

the amphiphile. In the case of lipid-like molecules, a P > 0.5 was

attributed to the favoured formation of spherical and cylindrical micelles.

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The situation 0.5 < P < 1 leads to less curved bilayers. Nevertheless it is

more appropriate for amphiphilic block copolymers to use the volume or

weight fraction of the hydrophilic block to describe their shape.30

1.3.2. Polymeric vesicles (polymersomes)

The most important representatives among the self-assembled

superstructures in this thesis are polymer vesicles, known as

polymersomes, which were generated by self-assembling amphiphilic

copolymers in dilute aqueous solutions, in a similar way to lipids.26,31

These vesicles are spherical objects with closed-block-copolymer

membranes in diameters ranging from 50 nm to approximately 10 μm,

depending on the chemical composition, the size as well as the

hydrophobic/hydrophilic ratio32,33 of the polymer blocks, the preparation

method and the preparation conditions. These polymer vesicles are

significantly more stable to lysis by classical surfactants than liposomes,

while preserving low immunogenicity.26,34 In addition, they are highly

impermeable compared to liposomes,34 can be stimuli responsive and

can be functionalised for targeting approaches.35,36 Various techniques

were used to generate polymersomes such as direct dissolution of

copolymers (direct dissolution method), addition of the copolymer

previously dissolved in an organic solvent to aqueous media (cosolvent

method), or hydration of a copolymer film (film hydration method).31

Depending on the intended application, polymersomes may be adapted to

specific sizes for particular routes of administration. The polymer

membrane is organised in the way that the hydrophilic part is exposed to

the inner and outer aqueous environments34 and serves to partition

aqueous volumes with different compositions and concentrations, due to

a selective permeability towards hydrophobic and hydrophilic molecules.24

The molecular weight of the used block copolymers directly reflects


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membrane thickness, which can be up to 30 nm.37 The high mechanical

polymersome membrane stability leads to a long-lasting retention of

payloads, ideally suited for technological applications. Beside the

possibility to encapsulate hydrophilic guest molecules, polymersomes can

simultaneously carry hydrophobic molecules in their membrane.38

Vesicular morphologies are illustrated based on AB diblock and ABA

triblock copolymer (Fig. 3).

Fig. 3 Polymersome formation based on AB-diblock copolymer in comparison to

ABA-triblock copolymer.

1.3.3. Polymersome preparation methods

The procedure to prepare polymersomes requires that the specificity of

the building blocks, the conditions for the active compounds in case of

nanoreactor (see chapter 1.4) preparation, the architecture etc. are

adequate for the intended application. Especially in medical applications,

when polymersomes are used in the human body, the safety issue

increases considerably.

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Only the relevant techniques applied in the framework of our project,

the film hydration technique and the direct dissolution method will be

further discussed.

The film rehydration method consists of two stages: Firstly, the

amphiphiles are dissolved in an organic solvent and subsequently the

solvent is evaporated to form an uniform, thin film on the surface of a

glass flask.39 Afterwards, the self-assembly process towards

macromolecular superstructures such as vesicles is initiated by the

addition of the aqueous solution, accompanied by additional forces such

as stirring, vortexing or sonication. The aqueous solution may contain

active compounds, which will result in their encapsulation in the aqueous

cavity or their incorporation in the membrane of the supramolecular

structures (e.g. vesicle membrane).

In the direct dissolution method, polymers are directly dissolved in the

aqueous solution, which may contain active compounds for nanoreactor

preparation, by extended agitation until the polymer is completely

hydrated.24

1.3.4. Characterisation methods of polymersomes

Polymersome characterisation starts with the detailed investigation of

the building blocks and continues with the characterisation of the

architectures with regard to their size, stability, flexibility, charge, etc.

When active compounds are involved, overall nanoreactor activity needs

to be determined.

Polymersome size and size distribution24,40 (polydispersity index), the

aggregation concentration,41 morphology42 and change of size or shape

with variation of pH, temperature or detergent addition43,44 can be

determined by light scattering (static and dynamic light scattering)

experiments. Parameters such as weight averaged molecular mass,


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radius of gyration, hydrodynamic radius as well as information about

particle – particle and particle – solvent interactions can be deduced.42

For direct visualisation of self-assembled structures most commonly

microscopy techniques, such as optical microscopy, laser-scanning

microscopy, or electron microscopy are used. These techniques allow to

extensively characterise structures in a broad range from a few

nanometres (super resolution microscopy such as STED (Stimulated

Emission Depletion) or STORM (Stochastic Optical Reconstruction

Microscopy)) to a few micrometres.45

The simplest method to visualise the assemblies under ‘physiological’

conditions is offered by optical microscopy, but the limited resolution –

max resolution corresponds to the half of the used wavelength (around

200 nm) – of these devices is a disadvantage compared to electron

microscopy. Therefore, only large assemblies such as giant liposomes or

giant polymersomes can be observed by optical microscopy.

Electron microscopy techniques, including transmission electron

microscopy (TEM), scanning electron microscopy (SEM), cryo-TEM and

cryo-SEM, provide higher resolution and allow to visualise self-assembled

superstructures and nanoreactors.46-48 Thickness and structure of a

vesicle membrane and their phase behaviour can be observed by cryo-

TEM, cryo-SEM and the freeze-fracture technique.49

Fluorescence microscopy is established for observation of

fluorescently labelled nanocompartments, which contain fluorescent dyes

or fluorescent proteins. This method is used to study polymer assemblies

with respect to dye encapsulation behaviour or dye binding properties,50-53

and to investigate cellular uptake of polymersome nanocarriers or

nanoreactors. Furthermore, activity of nanoreactors after cellular uptake

can be proven by visualisation of fluorescent products in real-time.

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Confocal laser-scanning microscopy (CLSM) is a special kind of

fluorescence microscopy, where the specimen is point-wise irradiated and

its fluorescence emission is point-wise measured to create an image. The

main difference is the use of a confocal aperture, the pinhole, which is

adjustable and determines how fluorescence light coming from non-

focused planes is detected. This allows to image small sections and to

reconstruct 3D images containing different focus planes.

Fluorescence correlation spectroscopy (FCS) is used to investigate the

dynamic properties of fluorescent molecules such as dyes,

macromolecules and nanoparticles.54 The experimental technique is

based on monitoring fluorescence intensity fluctuations in a small

observation volume – a confocal volume. A correlation analysis of the

fluctuations yields information on the diffusion coefficients and times,

population distributions, concentrations, etc. It is also possible to

determine the size and quantify the number of encapsulated fluorescent

dyes or proteins in nanocontainers.51,52,54 The technique is widely used for

studies concerning encapsulation/entrapment/binding of fluorescent

molecules in/on polymer assemblies in order to determine the sensitivity

and stability of nanoreactors.46,51,55

1.4. Concept of nanoreactors based on amphiphilic block copolymers

In this thesis, a nanoreactor is defined as a polymersome

encapsulated with an active compound (for example enzymes, proteins,

enzyme-mimics) and rendered permeable for substrates/products by the

insertion of membrane proteins or ion channels in the polymersome

membrane (Fig. 4).

The concept of nanoreactor originates from the compartmentalisation

strategy in nature, where compartments are used to separate biological


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processes. Prominent examples in cells are organelles such as the

endoplasmic reticulum, the Golgi apparatus, mitochondria, peroxisomes,

the nucleus, etc., where biological molecules are produced, transported,

detoxified, stored or disposed of. The advantage of the

compartmentalisation strategy is that different biological processes can

simultaneously run without interfering each other due to portioning of

biological tasks. It further allows the protection of sensitive biomolecules

or provision of adapted conditions, while communication (exchange of

signals) enables complex cascade reactions. Similar to cell

compartments, nanoreactors are regarded as nanometre-sized reaction

compartments/spaces used for chemical and biological reactions.

Fig. 4 a) Polymersomes containing active compounds inside their aqueous cavity. b)

Polymer membrane, which is intrinsically permeable. c) A polymer membrane with

an inserted channel protein. Adapted with permission from Ref. 11 Copyright 2011

American Chemical Society.

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The concept of nanoreactors implies that the active compounds are

kept permanently in its aqueous cavity or in its membrane. This

compartmentalisation strategy has also the dual ability: the protection of

the content from proteolytic attack and providing a reaction space for in

situ activity.11 Nanoreactors were used to monitor11 or detoxify51,56

substances, to produce drugs and were applied for enzyme replacement

therapy.57

In medical applications, an increased efficacy of nanoreactors is

desirable compared to unspecific drug administration, which could be

realised by their site-specific action. Site-specific action can be triggered

by a specific targeting, which can be put into practice by functionalisation

of the nanoreactor`s surface with, for example, peptides, proteins,

antibodies or chelating agents to generate molecular recognition

points.52,53,55 Based on the highly specific molecular recognition, surface-

functionalised nanoreactors were able to act at specific locations.47,58,59

Also stimuli-responsive nanoreactors were generated, which can be

predetermined on or off “on demand” by different stimuli such as physical

(light, temperature, mechanical, magnetic, etc.), chemical (solvent, pH,

ionic strength) and biological (receptor, enzyme) triggers.60

1.4.1. Requirements for nanoreactors used in medical applications

In case of nanoreactor preparation, the proper combination of active

compounds with the polymersome architecture is needed (in the aqueous

cavity of the polymersome or in the polymersome membrane).

Nanoreactor functionality depends on the activity of the active compound,

which is specific and depends on the preparation method, the pH, ionic

strength, temperature and traces of solvents.10 Therefore, nanoreactor

preparation requires the preservation of the activity of the active

compound starting with the choice of the building blocks, which are


17

treated with organic solvents. These solvents, even in traces, could have

considerable effects on the activity of the biological compound. When

using a preparation method, the use of organic solvents should be

minimised or entirely organic solvent free preparation methods should be

used.24

Medical applications differ if nanoreactors are meant to be used inside

a cell as an in vivo application for diagnostics or therapeutics, or as ex

vivo, when nanoreactors are used as medical sensor for diagnostics.

When a nanoreactor is used outside a body, nanoreactor performance

with regard to ideal working conditions (pH, ionic strength, temperature,

etc. for the encapsulated, active compound is aspired solely. On the other

hand, nanoreactors applied in vivo have to fulfil complex requirements,

which belong to the safety standards provided by the U.S Food and Drug

Administration (FDA) and the European Medicines Agency (EMA). This

guarantees that the in situ biological activity of encapsulated active

molecules is ensured,2,61 the integrity and activity of the nanoreactor is

preserved in biological conditions and that the nanoreactor and its

components (nanoreactor building blocks and the active molecules) are

biocompatible and biodegradable25,56,62 and do not accumulate in the

body.

The size and morphology of a nanoreactor is crucial for its cellular

interactions. In this respect, nanoreactors of around 200 nm in diameter

have shown to be uptaken by cells.47,56 Therefore, nanoreactor

preparation methods need to be aimed to this size range. Preparation

methods also play an important role for preserving the biological activity

of the active species. Hence, solvents, which potentially lower enzymatic

activity, must be avoided.2 Also the chemical constitution and properties

of the nanoreactor system such as charge flexibility and density of the

building blocks can influence the activity of the active compound. Stability

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and integrity of the nanoreactors avoid the release of potential toxic

substances in the body. Nevertheless, nanoreactor membrane

permeability is desired for substrates and products. The efficacy of the

nanoreactor is enhanced by nanoreactors having stealth-like characters.

1.4.2. Active compounds supporting nanoreactor functionality

Which active compounds were used for nanoreactor engineering?

Most commonly, proteins, enzymes, enzyme-mimics, or photoactive dyes

have been entrapped in the nanoreactor compartments.10,25,60

Proteins are key molecules in cell activities. They catalyse metabolic

reactions, transcribe and replicate DNA, are stimuli responsive, transport

oxygen, etc. For example, haemoglobin was encapsulated in

polymersomes with a dual functionality of oxygen transport and

detoxification of peroxinitrite.63

A special class of proteins are enzymes, which specifically and

efficiently catalyse biological reactions to sustain life. They contain either

ion metal groups or organic functional groups, which can accelerate

metabolic reactions up to the diffusion-limit. Prominent catalytic

mechanisms were transition-state binding, entropic control of reactions,

acid-base catalysis, etc.

Enzyme-mimics were developed as artificial enzymes in order to

understand the functionality of biological counterparts in more detail, to

generate artificial enzymes with improved functionalities62 or to adopt

functions of enzymes.64

Also, photoactive dyes were encapsulated in nanoreactors to generate

reactive oxygen species for applications in cancer therapy.60 In this

context, encapsulation is important in order to reduce toxicity of the

photoactive dyes, while their efficacy can be increased due to application

of a local high dosage.


19

1.4.3. Incorporation of membrane proteins and ion-channels in amphiphilic

block copolymer polymersome membranes

The low permeability of the membrane of polymersomes towards a

great variety of molecules such as substrates requires that the active

compounds in the aqueous cavity of the polymersomes must be made

accessible. A strategy therefore is to permeabilise the membrane either

by using substrate-permeable membranes65 or by inserting membrane

proteins that serve as “gates”.66-68 Even though the dimensions of

synthetic membranes (>10 nm) exceeds the dimensions of membrane

proteins, functional incorporation in the membrane is feasible.

Membrane proteins, such as the aquaporin Z (AqpZ), LamB, TsX or

complex 1, are specific transporters,66,67,69,70 while OmpF or FhuA form

unspecific channels.68,71 The membrane proteins can tune the

permeability of the membrane in a selective manner when specific

transporters are inserted. In this thesis, the membrane protein OmpF and

the ion-channel gramicidin were used to render the polymersome

membrane permeable for substrates or trigger factors.

Fig. 5 Top view of OmpF. Figure generated from the protein database:

http://www.rcsb.org.

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OmpF

The outer membrane protein F (OmpF) is isolated from E. coli cultures

in quantities of tens of mg/mL cultures72 and is stable in conditions of up

to 70 °C in 2% sodium dodecyl sulphate.73 Dissolving OmpF in ethanol,

for example for membrane incorporation, has no influence on its activity

or stability. OmpF (Fig. 5) allows the passage of molecules up to a

molecular weight of 600 Daltons (Da).74 It has a beta-barrel structure, as

most of the channel proteins,74 which secondary structure has increased

stability compared to α-helical membrane proteins.

Functional incorporation of OmpF in PMOXA-PDMS-PMOXA

copolymer membranes was realised by Meier et al.75 As shown in Fig. 5,

OmpF is composed of three identical subunits, whereby the monomers

are mainly build by a 16-stranded antiparallel β-sheet barrel.76

Gramicidin

In contrast to OmpF, which unspecifically allows the passage of

molecules, gramicidin is an ion-channel, specific for monovalent cations.

Gramicidin is a polypeptide antibiotic, which is produced by Bacillus

brevis. It consists of 15 hydrophobic amino acids that are neither charged

nor polar with the common following sequence: formyl-L-val-gly-L-ala-o-

leu-L-ala-o-val-L-val-o-val-L-trp-o-leu-L-xxx-D-leu-L-trp-D-Ieu-L-trp-

ethanolamine, where xxx is tryptophan in gramicidin A, phenylalanine in

gramicidin B, and tyrosine in gramicidin C.77

Consequently, gramicidin is almost insoluble in water, but is soluble in

many apolar solvents, phospholipids and alcohols.

Due to its small size, gramicidin can adopt a variety of different

conformations. The two main conformations were determined as a helical

dimer or a double helical structure (Fig. 6).77 The helical dimer consists of


21

two monomeric gramicidin helices, which are associated with each other

N-terminus to N-terminus.

Fig. 6 Two main conformations of gramicidin pores are (A) a helical dimer (this

form leads to channel formation) or (B) a double helical structure (pore formation).

Reprinted with permission from Ref. 77 Copyright 1990 by Annual Reviews Inc.

The association between the two monomer- helices is stabilised by six

H-bonds. The dimer formation (this conformation is around 25 Å in length)

is crucial to cross a membrane (e.g. a lipid- bilayer) because one

gramicidin molecule alone would be too short to span the membrane.77

The channel, which is formed by the gramicidin helical dimer, is pervious

for monovalent cations such as H+ and K+ and has a diameter of 4 Å. In

nature, gramicidin serves as a defending mechanism against other

microorganisms. It has the ability to incorporate into lipid membranes,

rendering them permeable for ions thereby destroying the electrochemical

gradient, which is necessary for ATP production.

1.4.4. Engineering artificial organelles as cell implants based on nanoreactors

Due to the inherent advantageous properties of nanoreactors, their

development is highly directed towards applications inside the body. This

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could be either in the blood stream or in a bio-inspired approach by

mimicking cell organelles.2,47,61,62,78 Such an approach of developing "cell

implants" has large potential in the repair of cell dysfunctions directly at

the source, responsible of many disease such as Parkinson`s disease,

amyotrophic lateral sclerosis (ALS), Alzheimer, cancer, etc. This new

strategy will have an enormous impact on personalised medicine and in

the design of artificial cells to build more complex synthetic systems. To

date, only few examples of nanoreactors used as artificial organelles are

known, which can be explained by the complex requirements for their

design and preparation. Nevertheless, until now, the few claimed artificial

organelles even do not perform a biologically relevant task. Nanoreactors

must have appropriate sizes or are tagged with cell-targeting moieties for

enhanced cell-uptake. In addition, the activity of entrapped/encapsulated

enzymes must be maintained in situ and the nanoreactor must be stable

in order not to break under different environmental conditions, such as

low pH as present in lysosomes.

1.5. Concept of nanocarriers for drug/protein delivery based on amphiphilic

block copolymers

The concept of nanocarriers based on polymersomes emerged from

the desire to deliver active components such as drugs at high dosage to

specific locations. This was realised by the encapsulation (in the aqueous

cavity) and/or entrapment (in the membrane) of payloads (drugs,79,80

DNA,81 proteins,82,83 or RNA84) in the compartments and its directed

release.

Encapsulation efficiency plays a crucial role in the efficacy of the

payloads, especially when high dosages of drugs were needed. Different

attempts to increase the encapsulation efficiency, for example by

complexation of the encapsulated compounds85 or by optimisation of the


23

copolymer block lengths46 were performed. In addition, it is also important

that the nanocarriers can "detect" the target location for payload release.

This is achieved by a targeting approach, which in combination with

triggers, such as pH, temperature or light can efficiently control the

release of the active components. Nevertheless it is still challenging to

precisely control the release over time and it is unknown that payloads

are released in the right place of action.

1.6. Relevance of reactive oxygen species (ROS) in oxidative stress

In certain pathologies, the regulation of reactive oxygen species

(ROS), which include the superoxide radical anion (O2-•), peroxynitrite

(ONOO–), the hydroxyl radical (OH-•) and hydrogen peroxide (H2O2) is

disordered. ROS are highly reactive molecules due to their unpaired

electrons. They are generated during the oxygen metabolism as a by-

product and play an important role in cell signalling and homeostasis.86,87

Oxidative stress occurs when the metabolic status of an aerobic organism

is imbalanced with ROS by overwhelming the cellular antioxidant defence

mechanisms to the disadvantage of the organism`s antioxidant defence

mechanism. Oxidative stress has been shown to contribute to aging88 and

to play a significant role in many disease including arthritis, Parkinson`s

disease, amyotrophic lateral sclerosis (ALS), cancer and AIDS.89-92 There

are different sources, where ROS are produced as shown in Fig. 7.

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Fig. 7 Overview of the source of reactive oxygen species and its impact on

biomolecules and biological processes.

Exogenous agents such as smog, ozone, pesticides, ionizing

radiation, xenobiotics etc. and a variety of endogenous processes, such

as the mitochondrial respiration, cytochrome P-450 detoxification

reactions, xanthine oxidase oxidation, phagocytic oxidative bursts, and

peroxisomal leakage can generate significant amount of ROS. This leads

to damage, inactivation and modulation of biological molecules, such as

proteins, carbohydrates, lipids, and nucleic acids and of cellular events

due to the activity of ROS (Fig. 7).

Dramatic effect on suitable adaption of metabolic processes had to

occur due to the availability of oxygen in the course of evolution. Amongst

others, the development of antioxidant defence mechanisms to regulated

ROS was essential for survival.


25

1.6.1. Natural defence mechanism

Superoxide radicals and related hydrogen peroxide are continuously

produced during the mitochondrial respiratory as a result of univalent

oxygen reduction by Complex I.93 But also in peroxisomes, where the

fatty acid α-oxidation, the β-oxidation of very long chain fatty acids, the

catabolism of purines, and the biosynthesis of glycerolipids and bile acids

occurs, reactive oxygen species can be formed.94 The natural defence

mechanisms are able to reduce reactive oxygen species to tolerable, non-

toxic amounts due to their toxic effect at concentration above the

threshold produced during the metabolism or necessary for signal

transduction. Antioxidant substances are part of the defence mechanism,

which are able to minimise or prevent substrate oxidation. These can be

for example enzymes such as superoxide dismutase (SOD), catalase

(CAT), both found in peroxisomes, peroxidases (e.g. lactoperoxidase or

horseradish peroxidase), glutathione peroxidase and transferases,

quinone reductases or small molecular weight substances such as

glutathione, ascorbic acid, vitamin C and E, uric acid, carotenoids, etc.

able to act as antioxidants.

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Fig. 8 Schematic overview of ROS homeostasis occurring in peroxisomes. Specific

transporters (e.g. ABCD1) transport various peroxisomal metabolites such as

VLCFA across the membrane and most of the peroxisomal matrix proteins are

imported in a PTS1/Pex5p-dependent manner. Peroxisomal oxidases (light purple)

are responsible to break down the substrates so that the hydrogen extracted from

the substrate is directly transferred to O2. Peroxisomal antioxidant enzymes (light

blue) convert the generated H2O2 to H2O and O2. A by-product of the xanthine

oxidase (XO) activity is O2•–

, which is immediately reduced by manganese

superoxide dismutase (MnSOD) and copper-zinc-superoxide dismutase

(Cu/ZnSOD). Others, such as Mpv17 and M-LP (dark blue) are involved in the

regulation of peroxisomal ROS metabolism, even though it is not clear if they are

localized in the peroxisome. Reprinted with permission from Ref. 94. Copyright

2009 WILEY-VCH Verlag GmbH & Co. KGaA.

The importance of ROS detoxification is illustrated in the schematic

overview of ROS homeostasis occurring in peroxisomes, as shown in Fig.

8.94 It proceeds as a cascade reaction, where SOD detoxifies the O2•–


27

radicals to hydrogen peroxide, shown in equation 2.

(2)

The toxic character of H2O2 implies its further decomposition to

harmless products, water and O2, which is made either by CAT,

glutathione peroxidase, peroxiredoxin or PMP20, which also belongs to

the peroxiredoxin family.95 CAT detoxifies H2O2 in a catalactic reaction

(equation 3).

(3)

The mechanism, where glutathione peroxidase is involved, removes

H2O2 by the oxidation of glutathione (GSH) to its oxidised form,

glutathione disulphide (GSSG) (equation 4).

(4)

The peroxiredoxin (PRX) system uses thioredoxin (TRX) to detoxify

H2O2. The following reaction mechanism is proposed: (equation 5)

( ) ( ) (5)

Due to the inactivity of PRX (oxidised), it needs to be restored to its

reduced form by TRX, shown in equation 6.

( ) ( ) ( ) ( ) (6)

TRX is a polypeptide especially occurring in the endoplasmic reticulum

and in mitochondria. It contains a dithiol-disulfide active site. Its reduced

state is obtained by flavoenzyme thioredoxin reductase.

1.6.2. Antioxidant therapy

The aim of the antioxidant therapy is to counteract the functional

failure or the overcharge of natural antioxidant mechanisms by providing

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antioxidants. These are able to reduce the harmful effects of ROS and

thus prevent or cure oxidative stress and related diseases. Early

antioxidant therapies made use of antioxidants known from the natural

defence mechanism, such as SOD or CAT.96 But also vitamins, such as

vitamin A, vitamin C, or vitamin E were used to reduce the amount of

ROS.97 Nevertheless, the short half-life in blood plasma and difficulty for

efficient cellular uptake of antioxidant substances lower the efficacy of an

antioxidant therapy. For example it is known that SOD and CAT were

inadequately delivered to targeted cells.98,99 A strategy to increase uptake

behaviour was for example by conjugation of antioxidant enzymes with

antibodies to improve their biocompatibility.99,100 The risk is that

conjugation of enzymes with other molecules can dramatically lower the

activity of the enzymes.101

Nevertheless, both the use of antioxidant enzymes, as well as the use

of vitamins in order to efficiently treat conditions of oxidative in vivo did

not yield convincing benefits yet.

1.7. Concept of molecular recognition

Molecular recognition allows specific biological interactions by non-

covalent bonds such as hydrogen bonds, hydrophobic interactions, metal

coordination etc. and supports instant response in different processes.

Molecular recognition is distinguished between static molecular

recognition and dynamic recognition. Static molecular recognition can be

described as a key-lock system (Fig. 9A), where the key exactly fits into

the lock. Dynamic molecular recognition implies on the contrary that a

binding of a first guest molecule to the host allows a second molecule via

a conformation change to fit (induced fit) (Fig. 9B). Hence, in a positive

allosteric system, the binding of the first guest molecules increases the


29

binding affinity towards the second guest, while in a negative allosteric

system, the first guest molecule decreases the binding affinity towards the

second guest.

Fig. 9 Different models of substrate binding to enzymes: A) In the lock-key model,

the correctly sized and shaped substrate binds to the active site of an enzyme in

order to undergo an enzymatic reaction. B) In the induced fit model, the substrate

determines the correct shape of the active site of the enzyme, in order to process the

substrate to the product.

Technical realisation of the molecular recognition concept resulted in

the principle of key-lock systems or the use of affinity tags and is widely

applied in the purification102,103 and immobilisation20,23,104,105 of

biomolecules, protein labelling,106,107 targeting approaches,108 etc.

1.7.1. Molecular recognition strategies in nature

Molecular recognition in biological systems helps molecules to self-

assemble, organise, recognise or communicate and to achieve biological

function. Natural recognition models are for example the

antigen−antibody,109,110 receptor−ligand systems (the nicotinic

acetylcholine receptor or the folate receptor),111 DNA−protein

assemblies,112 or RNA –ribosomes.

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1.7.2. Targeting strategies

In order to specifically target nanocarriers or nanoreactors, the natural

principle of molecular recognition, which allows identification of patterns

to be recognised by each other, can be applied to synthetic systems. It is

achieved by functionalisation of the polymersome surface to build specific

recognition points, which is presented in the following section.

Metal-functionalised polymer membranes

Functionalisation of amphiphilic diblock copolymers with metal-based

recognition sites that can self-assemble to polymersomes allow

production of metal-functionalised polymer membranes. In these

supramolecular assemblies, the free coordination points of the metal

centre interacts with biomolecules by replacing the solvent molecules and

rearranging its geometry.55 Prominent ligands that are able to be

recognised by peptides and proteins when complexed with metals such

as copper or nickel are iminodiacetato (IDA), bis(2-pyridylmethyl)amine

(BPA), diethylenetriamine (DIEN), nitrilotriacetic acid (NTA) or tris

nitrilotriacetic acid (trisNTA), tris(2-pyridylmethyl)amine (TPA), and tri(2-

aminoethyl)amine (TREN).55,113 This approach finds application in the

selective binding of biological molecules for functional and oriented

immobilisation,114 and can be successfully used for 2D and 3D protein

immobilisation. Polymersomes, functionalised with specific molecular

moieties on the outer surface, such as biotin, antibodies, or metal

complexes,21,23 allow for cellular uptake, protein binding or can be used to

be immobilised on solid supports.52

1.7.3. Metal-chelation: NTA and trisNTA

Metal-chelation describes the ability of molecules or ions to bind to

metal sites and implies that the binding of a polydentate ligand to the


31

metal centre consists of at least two coordination bonds. The metal

complexes formed with these ligands have a coordination sphere that

consists of strongly binding atoms of the ligand and weak binding solvent

molecules. The biomolecule interacts with the metal ion as it replaces the

solvent molecules and rearranges the geometry. This binding mechanism

confers the advantage of stable immobilisation of the biomolecule,

similarly to nature.115 In this respect, NTA- and IDA-metal complexes

have been used to design synthetic receptors that bind peptides or

proteins under physiological conditions.113,116 The NTA ligand is widely

applied in immobilised-metal affinity chromatography.117 It coordinates for

example the Ni2+ with four valences as shown Fig. 10.

Fig. 10 Model of the interaction between residues in the His-tag and the metal ion in

tetra-pentadentate NTA ligand. Reprinted with permission form Ref. 117.

Copyright 2009 Elsevier Inc.

In this molecular configuration, two additional valences are available to

be coordinated to the imidazole rings of histidine residues. In a further

development, increased binding capability was achieved by using trisNTA

moieties for protein immobilisation studies. The trisNTA molecules (Fig.

11b, here complexed with CuII) feature three NTA (Fig. 11a) moieties, all

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complexed with a metal ion, which are simultaneously involved in the

ligand-binding process.

Fig. 11 Schematic formula of a) CuII

−NTA complex and b) CuII

− trisNTA. X

represents solvent molecules or external binding atoms of other molecules.

Reprinted with permission from Ref. 55. Copyright 2012 American Chemical

Society.

1.7.3. His-tags and His-tagged molecules

His-tagged molecules can be used not only for immobilisation or

purification purpose of proteins but also in order to study molecular

recognition interactions. These molecules most commonly consist of a tag

moiety with at least six consecutive histidines covalently bound. Other

reported His-tag sequences were summarised in a review of immobilised-

metal affinity chromatography.117 The tag moiety has been shown to

specifically bind to metal binding sites by a shift of the

association/dissociation equilibrium constant to the site of association as

a result of the known affinity of histidines to transition metals, such as

Zn2+, Cu2+, Ni2+, or Co2+.115 There, typical dissociation rates of different

systems were determined to range between 10-6 and 10-9 M. The


33

system`s properties, such as the used medium, buffer solution, pH, ligand

density or concentration or accessibility can have an influence on the

binding behaviour. As an example, the oligohistidine sequence consisting

of six histidines efficiently interacts with NiII−NTA complexes with a

binding affinity in the subnanomolar range.107,118,119 When the number of

histidines on each tag was increased, for example, 10 histidines instead

of 6 histidines) a decreased metal binding affinity was observed.115 His6-

tag affinity to different transition-metal ions was predicted by molecular

dynamic simulations, including Cu2+, Fe3+, Ni2+, and K+ ions,. The results

indicated that the His6-tag exhibits the strongest binding affinity for Ni2+

and Cu2+ ions.120

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35

2. Combat superoxide anion radicals by antioxidant nanoreactors

A key challenge in life and nano-sciences is to fuse biological entities,

such as enzymes, with synthetic materials, for example block copolymers

in order to create new, complex synthetic biological devices. Combination

of specificity and efficiency of biological molecules with robustness and

possibility of tailoring of polymeric materials allows the design of efficient

mimics of living entities, or of electronic-inspired systems, on the nano-

scale domain. These out-perform conventional chemical or biological

approaches to generate revolutionary, highly-specific communicating or

processing systems, such as biosensors, signal amplifiers, electron-

transfer devices, or nanoreactors.1,25,121 Enzymatic activity can be

improved and enzymatic pathways customised by combining synthetic

with biological materials, as has been shown by trypsin encapsulation in

PS-b-PAA block copolymers122 or by reconstitution of ubiquinone

oxidoreductase in copolymer membranes.69 However, a real challenge is

related to the preservation of the enzyme activity as well as its integrity

when combined with polymeric systems. This renders the situation more

complex than in bulk conditions, and requires mild procedures of

generating the hybrid systems, as well as to establish the polymer/protein

interactions, and their effect on enzyme structure and activity.

Recently such bio-synthetic materials were successfully applied for

local bio-conversion on surfaces,53 and inside living cells.61 Here we plan

to extend these concepts to the detoxification of reactive oxygen species

(ROS), including the superoxide radical anion (O2•-), peroxynitrite,

hydroxyl radical, and hydrogen peroxide, those natural production is

dramatically increased during oxidative stress.123 Oxidative stress has

been reported to play an important role both related to the toxicology of

inorganic nanoparticles,124 and in the pathogenesis of many diseases,

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such as arthritis, Parkinson’s disease, amyotrophic lateral sclerosis,

cancer, or AIDS.89,90,125

2.1. Motivation and concept

Many attempts to detoxify ROS by direct antioxidant enzyme

administration were undertaken, but no conclusive data was obtained.

SOD, for example, was quickly eliminated from the blood stream.98 In

addition, attempts to modify SOD in order to improve delivery were tried.

To this end, SOD was covalently modified with poly(ethylene glycol)

(PEG)126 or encapsulated in liposomes127,128 and in polymeric

microspheres.129 But none of these attempts could improve SOD delivery

significantly.

A promising attempt was the design of antioxidant nanoreactors

encapsulating SOD inside oxygen permeable polymeric vesicles. The

enzyme efficiently catalysed the reaction of superoxide radicals to

generate hydrogen peroxide.51 However, hydrogen peroxide as a final

product is well known to still contribute to oxidative damage.130 Therefore,

in order to detoxify superoxide radicals and related harmful hydrogen

peroxide, similar to cellular antioxidant mechanisms, an intricate system

must be engineered.

Here, we use this concept in order to engineer advanced nanoreactors

based on the encapsulation of two different antioxidant enzymes,

superoxide dismutase (SOD) and lactoperoxidase (LPO) or catalase

(CAT), in polymersomes for detection and detoxification of reactive

oxygen species (ROS). The encapsulated antioxidant enzymes act

simultaneously in situ while protected from proteolytic attack.15,41


37

Fig. 12 a) Specialised enzymatic cascade reaction inside polymeric nanovesicles for

detection and detoxification of superoxide radicals. Two different types of enzymes

act inside the polymersome in a cascade reaction with substrates (D) and products

(F) to detoxify reactive oxygen species (A). Substrates, products, and reactive

oxygen species penetrate the polymeric membrane due to the insertion of protein

channels (B); b) Schematic illustration of the reaction mechanism of the SOD/LPO

cascade reaction inside a polymersome; c) Chemical reactions allowing the detection

and detoxification of superoxide radicals: 1) generation of O2-•; 2) reactions

involving SOD; 3) reactions involving LPO. Adapted with permission from Ref. 56.

Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

The selected enzymes catalyse the same reaction in nature.

Superoxide radicals can diffuse through the polymer membrane and

reach the enzymes, where they were reduced in a cascade reaction to

harmless reaction products H2O and molecular oxygen (Fig. 12b).

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In order to observe the detoxification process, i. e. the detection of

superoxide radicals and testing the antioxidant activity inside cells,

membrane proteins were needed in order to allow the passage of

substrates and products of LPO through the polymer membrane, which is

highly impermeable to small molecules.56 This was realised by the

incorporation of the outer membrane protein F (OmpF) in the vesicle

membrane to permit passive diffusion of molecules of sizes smaller than

600 Da.74 OmpF allowed that a second substrate for LPO, amplex red,

was able to penetrate the aqueous cavity of the vesicles, which was

converted in situ to a fluorescent product, resorufin, so that the activity of

the enzymes could directly be observed.

2.2. Design of an enzymatic cascade reaction to fight oxidative stress

The cascade reaction is initialised by SOD, which catalyses the

reduction of superoxide radicals, generated for test purpose in the initial

reaction by xanthine/xanthine oxidase (Fig. 12c), to hydrogen peroxide at

high efficiency. The reaction rate remains unchanged at physiological pH,

the condition used here, and appears not to be affected by pH variation in

range between pH 6 and 9.131 In the second step of the cascade reaction,

hydrogen peroxide is decomposed by LPO or CAT in presence of one of

its specific substrate, amplex red, which is converted to a fluorescent

product resorufin that can easily be detected (Fig. 12c). LPO was chosen

as a second enzyme due to its high activity at physiological pH – 80% of

its activity is preserved,132 which makes it compatible to SOD. In addition,

the ability to use a second substrate for LPO to generate a fluorescent

product in the end of the cascade reaction allows detailed

characterisation of the enzyme kinetics in bulk, in the nanoreactor and

when used as artificial organelle.


39

In order to further optimise the cascade reaction, the use of CAT

instead of LPO as the second enzyme was considered. CAT plays an

important role in the ROS homeostasis of peroxisomes and is therefore a

promising enzyme in order to detoxify hydrogen peroxide.

2.3. Design and characterisation of antioxidant nanoreactors

We engineered antioxidant nanoreactors comprising of membrane

protein gated polymersomes, which are able to detoxify superoxide anion

radicals in the aqueous cavity via a cascade reaction. The cascade

reaction is based on the activity of Cu/Zn-SOD, which is the first enzyme,

and LPO or catalase (CAT) as the second enzyme.

To provide the reaction cavity, polymersome superstructures were

generated from the amphiphilic triblock copolymer poly(2-

methyloxazoline)-b-poly(dimethylsiloxane)-b-poly(2-methyloxazoline)

(PMOXA-PDMS-PMOXA) by self-assembly in aqueous solution at

physiological condition. The generated vesicles were mechanically very

stable,133 and do not comprise of structural defects as known from

liposomes. The used polymer membrane is highly permeable to

superoxide anion radicals, but impermeable to higher molecular weight

molecules such as saccharide ions or water.67 The functional

incorporation of OmpF in the polymersome membrane allows only a

passive diffusion of molecules up to 600 Da74 and thus is appropriate for

the use with a second substrate for LPO, amplex red, which is converted

in the presence of hydrogen peroxide, to a fluorescent product, resorufin.

This in turn simplifies the detection of the superoxide anion radical and

hydrogen peroxide detoxification process.

Based on the chemical nature and the hydrophobic-to-hydrophilic ratio

of the used PMOXA12-PDMS55-PMOXA12 copolymer, polymersome

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preparation was made using the direct dissolution method. This method

provides mild conditions so that the integrity and activity of different

enzymes encapsulated in nanoreactors were preserved.56,63

Fig. 13 A) TEM micrograph of empty PMOXA-PDMS-PMOXA polymersomes.

Scale bar = 200 nm; Light scattering of empty polymersomes: B) First order fit of

DLS data; C) Guinier plot representation of SLS data. Reprinted with permission

from Ref. 78. Copyright 2013 American Chemical Society.

Via this preparation method, homogeneous, empty PMOXA-PDMS-

PMOXA polymer vesicles with a diameter of 200 ± 40 nm were formed in

PBS buffer, as confirmed by TEM (Fig. 13A) and dynamic and static light

scattering (Fig. 13B and C).

Membrane permeability was adjusted according to an increase of

permeability while keeping the polymersomes intact. An OmpF

concentration of 50 µg/mL proved optimal for membrane permeability,

and did not affect membrane stability.70


41

2.3.1. Optimised encapsulation of individual enzymes in polymersomes

In order to work the best condition out to engineer efficient co-

encapsulated antioxidant nanoreactors, characterisation and optimisation

of each encapsulated enzyme type must be evaluated individually. In this

respect, individual encapsulation efficiencies needed to be assessed –

taking care not to induce vesicle aggregation or to disturb the vesicle self-

assembly process.

Fig. 14 A) TEM micrograph of SOD-encapsulated PMOXA-PDMS-PMOXA

polymersomes. Scale bar = 200 nm; Light scattering of SOD-encapsulated

polymersomes: B) Second order fit of DLS data; C) Guinier plot representation of

SLS data. Reprinted with permission from Ref. 78. Copyright 2013 American

Chemical Society.

The different concentrations used for polymersome preparation did not

affect their morphology or size of the nanoreactors, as established by a

combination of light scattering and TEM for: SOD (Fig. 14), LPO (Fig.

15), and CAT (Fig. 16).

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Fig. 15 A) TEM micrograph of LPO-encapsulated PMOXA-PDMS-PMOXA

polymersomes. Scale bar = 200 nm; Light scattering of LPO-encapsulated

polymersomes: B) Second order fit of DLS data; C) Guinier plot representation of

SLS data. Reprinted with permission from Ref. 78. Copyright 2013 American

Chemical Society.

In order to measure and quantify the number of encapsulated

enzymes and to increase the encapsulation efficiency of enzymes per

polymersome, the enzymes were labelled with different fluorescent dyes

− AlexaFluor-488 for SOD, AlexaFluor-633 or dylight-633 for LPO, and

dylight-488 for CAT − and the efficiency was determined by a combination

of fluorescence correlation spectroscopy and fluorescence brightness

measurements.


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Fig. 16 TEM micrograph of CAT-encapsulated polymersomes. Scale bar = 200 nm.


Society.

Upon labelling SOD with AlexaFluor-488, a labelling efficiency of three

AlexaFluor-488 molecules per SOD was obtained, which was considered

for the calculation of the encapsulation efficiency. Furthermore, one

AlexaFluor-633 molecule or 2.7 dylight-633 molecules per LPO, and 1

dylight-488 molecule per CAT was achieved.

The molecular brightness of the enzyme-containing polymersomes,

recorded as counts per molecules (CPM), was divided by the CPM of

freely-diffusing labelled enzymes to determine the number of enzymes

per vesicle. This number was then used to calculate the encapsulation

efficiency in vesicles (EEv) based on the initial enzyme concentration by

dividing the number of enzyme molecules per polymersome by the

maximum number, which could be theoretically encapsulated inside a

vesicle via a statistical, self-assembly process of encapsulation. The

detailed encapsulation efficiency (EEv) calculation is shown in the

Appendix.

When SOD-AlexaFluor-488 was encapsulated in polymersomes, the

diffusion time D changed from 109 s, which corresponds to the free

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SOD-AlexaFluor-488, to a value of 3.5 ± 1 ms, which indicates that the

enzyme was encapsulated in vesicles (Fig. 17A).

Fig. 17 A) FCS autocorrelation curves and the simulation of: a) free AlexaFluor-

488; b) SOD-AlexaFluor-488; c) SOD-AlexaFluor-488-containing vesicles; B)

Comparison of the theoretical and experimental amount of enzymes encapsulated in

a polymer vesicle as a function of initial enzyme concentration: Theoretical

(squares) and experimental (dots) number of SOD encapsulated in 75 nm radius-

sized polymersomes. Reprinted with permission from Ref. 78. Copyright 2013


Preparing polymersomes with different initial amounts of SOD-

AlexaFluor-488 from 0.1 mg/mL to 0.5 mg/mL allowed the optimisation of

a maximum encapsulation efficiency of EEv of 25 ± 10% at an initial

concentration of 0.25 mg/mL (Fig. 17B). To rule unspecific binding of the

enzyme or of the labelled enzyme out, which would affect the calculated

number of encapsulated enzymes, control measurements using empty

polymersomes incubated with dye-labelled enzymes or with only the dye

were performed. In this case, no change of diffusion time was observed,

proving that neither AlexaFluor-488 nor SOD-AlexaFluor-488 interact to

empty polymersomes. Increasing the SOD concentration used for

encapsulation to 0.9 mg/mL caused solubility problems of SOD being the

limiting factor of further increase. The resulting high encapsulation

efficiency of SOD together with the high reaction kinetics


45

(kcat ≈ 10-9 M-1 s-1)131 were basic pre-requisites for efficient antioxidant

properties.

Similarly, encapsulation of LPO-AlexaFluor-633 in polymersomes was

characterised by a change in the diffusion time from 320 s,

corresponding to the free LPO-AlexaFluor-633, to 8.5 ± 1 ms, showing

successful enzyme encapsulation (Fig. 18A). In order to optimise LPO

encapsulation efficiency, differently concentrated enzyme solutions from

0.1 mg/mL to 0.5 mg/mL were used. The optimum EEv of 66 ± 33% was

reached with an initial LPO concentration of 0.25 mg/mL (Fig. 18B).


633; b) LPO-AlexaFluor-633; c) LPO-AlexaFluor-633-containing polymersomes; B)

Comparison of the theoretical and experimental amount of enzymes encapsulated in

a polymer vesicle as a function of initial enzyme concentration: Theoretical

(squares) and experimental (dots) number of LPO encapsulated in 75 nm radius-

sized polymersomes. Reprinted with permission from Ref. 78. Copyright 2013


Further increase of the concentration of LPO-AlexaFluor-633 used for

the encapsulation to 0.83 mg/mL led to the formation of aggregates

during the self-assembly process, characterised by FCS with diffusion

times of >30 ms and confirmed by TEM (Fig. 19A and B).

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633 (50 nM) and b) LPO-AlexaFluor-633-containing aggregates (0.83 mg/mL); B)

TEM micrograph of LPO-AlexaFluor-633 containing aggregates. Scale bar = 2 μm.


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The reason for aggregate formation is the unspecific binding, ob-

served, when free AlexaFluor-633 was incubated with empty polymer-

somes, which increased the diffusion time to 7 ms. When a high, initial

LPO-AlexaFluor-633 concentration was used for encapsulation, non-en-

capsulated labelled enzymes served as aggregation points on the outer

vesicle surface and induced aggregate formation. When LPO was

labelled with dylight-633 using the same high initial concentration, the

formation of aggregates could be avoided (Fig. 20).


47

Fig. 20 Fluorescence autocorrelation curves: FCS autocorrelation curves (line) and

the simulation (dashed line) of a) free dylight-633 (50 nM); b) dylight-633-labelled

LPO (50 nM) and c) empty ABA polymersomes incubated with dylight-633. Re-

printed with permission from Ref. 78. Copyright 2013 American Chemical Society.

Nevertheless, aggregate formation is not a limiting factor of the optimi-

sation of the encapsulation efficiency, because the optimum EEv was ob-

tained at significantly lower concentration. Therefore, all further experi-

ments were conducted with LPO-AlexaFluor-633 for nanoreactor-, and

later artificial peroxisome-characterisation.

The modular build-up of nanoreactors allows to exchange enzymes for

further adaption to specific functions. Instead of LPO, we used CAT as a

second enzyme to test, if the nanoreactor activity could be further

increased. CAT is differed from LPO, it detoxifies H2O2 in a catalactic

reaction without the necessity of additional co-substrates and it has a

higher molecular weight of 250 kDa.134 When CAT-dylight-488 was

encapsulated, FCS measurements resulted in diffusion times of 4 ± 1 ms,

which can be attributed to CAT-dylight-488-containing polymersomes

(Fig. 21c). On the other hand, a diffusion time of 340 s was

characteristic of the free, labelled-enzyme (Fig. 21a), whereas the

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diffusion time for the free dye was 44 s. No aggregates were formed for

all different CAT concentrations used during vesicle formation.

Fig. 21 Normalized FCS autocorrelation curves (lines) and the simulation (dashed

lines) of a) free dylight-488; b) CAT-dylight-488 and c) CAT-dylight-488-containing

PMOXA12-PDMS55-PMOXA12 vesicles. Reprinted with permission from Ref. 78.

Copyright 2013 American Chemical Society.

A maximal EEv of 16 ± 9% was calculated based on brightness

measurements, which is lower compared to LPO. This can be explained

by the higher molecular weight of CAT compared to LPO, which creates

problems for efficient encapsulation, as shown before with lipid

vesicles.135

2.3.2. Activity assay of individual enzyme species in solution and in

polymersomes

LPO activity of the free enzyme in solution and encapsulated in

polymersomes was determined by using an activity assay comprising of

amplex red, which is converted by LPO in presence of hydrogen peroxide

to resorufin, shown in Fig. 22A and B, respectively.


49

Fig. 22 A) Activity assay for LPO in free conditions with different concentrations of

amplex red: a) 3.75 µM; b) 1.88 µM; c) 0.94 µM and d) 0.47 µM. B) Activity assay

of: a) empty polymersomes (amplex red 0.5 µM); b) LPO encapsulated in

polymersomes without channel proteins (amplex red 0.5 µM) and LPO encapsulated

in polymersomes with channel proteins and with gradient of amplex red: c) 0.18

µM; d) 0.37 µM; e) 0.5 µM and f) 1 µM. Reprinted with permission from Ref. 56.

Copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA.

In order to measure enzyme kinetics, different substrate

concentrations of amplex red were used. As expected, polymersomes

containing LPO without OmpF gated membranes produced no resorufin

signal. On the other hand, LPO containing vesicles with gated

membranes clearly converted amplex red to resorufin in proportion to the

initial substrate concentration. This demonstrates that OmpF allows

substrates diffusing through the membrane.

Calculation of the kinetic constants for the LPO enzymatic reaction (KM

and Vmax) in free conditions compared to encapsulated conditions using

the Lineweaver-Burk equation136 gave information about the preservation

of enzyme activity. This showed that substrate affinity to LPO in

nanovesicles is slightly higher (KM value of 2.9 ± 0.1 µM) compared to

free conditions (KM value of 9.4 ± 0.3 µM). Higher substrate affinities in

encapsulated condition have already been reported,122 which results from

higher collision frequencies, due to the location of both enzyme and

substrate at the same interface. Comparison of the maximal reaction

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rates Vmax of the encapsulated enzyme (37.1 ± 7.1 µMs-1) with the

enzyme in free condition (132.3 ± 75.9 µMs-1) shows that it is lower in the

encapsulated condition, but still comparable to the free condition.

Fig. 23 LPO activity over time, when a) encapsulated in polymersomes and b) in

free condition. Reprinted with permission from Ref. 56. Copyright 2011 Wiley-VCH

Verlag GmbH & Co. KGaA.

The stability of encapsulated LPO was also compared to free LPO

over a time period of 60 days (Fig. 23); both systems were stored in dark

at 4 °C. After 60 days, LPO activity inside the polymersomes was still

approximately 70%, while the activity of LPO free in solution decreased to

40%, which could be a result of possible contamination with small

amounts of proteases of the free enzyme containing solution. This further

confirms that the aqueous cavity of the polymersomes provides

advantageous conditions for the enzyme in means of protection, which

was also reported for SOD containing polymersomes.51

During the optimisation of the antioxidant nanoreactor, we also

compared the enzyme kinetics of LPO-containing polymersomes to that

of CAT-containing polymersomes according to the activity assay

presented in Scheme 1. At comparable concentrations of LPO and CAT


51

in free condition adjusted to the concentration range expected to be

present in polymersomes, CAT activity was comparable to the activity of

LPO (Fig. 24A). CAT activity was also measured in CAT-encapsulated

polymer vesicles. The successful incorporation of the channel protein

OmpF, which allows passage of H2O2 and amplex red through the

membrane, led to a clear decrease in the fluorescence signal (Fig. 24B

b) compared to the condition in which no CAT-encapsulated polymer

vesicles were present (Fig. 24B a). Thus, CAT was encapsulated in an

active form.

Fig. 24 A) CAT activity assay based on an assay containing LPO (50 nM), H2O2

(3 µM) and amplex red (0.5 µM) to monitor CAT activity in free conditions using

different CAT concentrations: a) 0 nM; b) 10 nM; c) 50 nM; d) 100 nM. B) CAT

activity assay based on an assay containing LPO (10 nM), H2O2 (0.6 µM) and

amplex red (0.1 µM) to monitor CAT activity: a) without nanovesicles; b) in

nanovesicles with incorporated OmpF channel membrane. [CR] = count rate.


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Similar kinetics between peroxidases and CAT at low substrate

concentrations and under physiological conditions have already been

reported.137 But the result of the lower encapsulation efficiency of CAT

compared to LPO makes hydrogen peroxide degradation by CAT-

containing polymersomes less effective. The determinant of the enzyme

activity is in this case the encapsulation efficiency and not the specificity

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of the enzyme. Therefore, further experiments were performed using only

LPO as second enzyme.

2.3.3. Optimised co-encapsulation of SOD and LPO in polymersomes

Superoxide radical detoxification by the antioxidant nanoreactors

needs successful co-encapsulation of both enzyme types, SOD and LPO,

in polymersomes.

Fig. 25 Turbid solution containing SOD/LPO-co-encapsulated polymersomes.

(Sample after size-exclusion- and dye separation (dialysis) process.) Reprinted with

permission from Ref. 78. Copyright 2013 American Chemical Society.

The simultaneous co-encapsulation, using the optimised condition of

each individual enzyme type and self-assembly of the block copolymer

produced a turbid solution (Fig. 25). Co-encapsulation did not affect the

morphology or the size of the vesicles, as shown by TEM and light

scattering (Fig. 26A, B and C).


53

Fig. 26 A) TEM micrograph of SOD/LPO co-encapsulated polymersomes; Light

scattering data for SOD/LPO-co-encapsulated vesicles: B) Second order fits of DLS

data; C) Guinier plot representation of SLS data. Adapted with permission from

Ref. 78. Copyright 2013 American Chemical Society.

In order to relate the ROS detoxification efficacy with the quantity of

co-encapsulated enzymes, fluorescence cross-correlation spectroscopy

(FCCS) was used. Because the encapsulation of the enzymes inside

polymersomes is based on a probabilistic process due to the unspecific

inclusion of molecules during the self-assembly process in vesicles, the

encapsulation of two different types of enzymes can produce the following

scenario. Either only one type of enzyme is encapsulated, none of them

were encapsulated, or both types of enzymes were simultaneously

encapsulated. In order to quantify the co-encapsulation, FCCS is used,

which is based on monitoring the fluorescence intensity fluctuation of two

fluorescent species in overlaying confocal volumes. If the two species are

moving in a simultaneous way, e.g. when located inside the same

confinement, a cross-correlation can be calculated, which is proportional

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to the degree of simultaneous movement. This technique allows to

determine the degree of co-localisation of the fluorescently labelled

enzymes SOD and LPO in the same polymersome.

Fig. 27 FCS and FCCS of SOD/LPO co-encapsulated polymersomes: a)

autocorrelation curves of vesicles containing SOD-AlexaFluor-488; b)

autocorrelation curves of vesicles containing LPO-AlexaFluor-633, and c) Cross-

correlation curves of SOD-AlexaFluor-488 and LPO-AlexaFluor-633 co-

encapsulated in the polymersomes. Dotted lines are experimental curves. Reprinted

with permission from Ref. 78. Copyright 2013 American Chemical Society.

Successful co-encapsulation was obtained, as shown in the positive

cross-correlation curve of the FCCS measurement (Fig. 27c). Qualitative

analysis of the fluorescence brightness measured in each channel (Fig.

27a and b) of the FCS measurement revealed that an enzyme ratio of

SOD:LPO of 1:2 was present inside the polymersomes. Fluorescence

from the first species detected in the second channel and vice versa, also

known as cross-talk, was negligible (<0.5 kHz) in the FCCS experiment,

due to the well-separated fluorescence emission spectra of AlexaFluor-

488 and AlexaFluor-633. Calculation of the relative amplitude of the

cross-correlation curve in relation to the amplitude of the auto-correlation

curve allows calculating that approximately 22 ± 10% of the

polymersomes contained simultaneously both enzymes. This is close to


55

the value of a theoretical calculation, based on the self-assembly process

of vesicle formation during individual enzyme encapsulation.

2.3.4. Multi-enzyme kinetics in a solution that simulates polymersome conditions

In order to carry out complex reaction as efficiently and in accordance

with nature, specific enzyme functionality in multi-enzyme systems needs

to be maintained. Based on the result of the FCS/FCCS quantitative

analysis, activity tests of the SOD/LPO cascade reaction in solution under

conditions simulating the concentration ranges of enzymes encapsulated

in polymersomes were performed.

Fig. 28 A) Cascade reaction in free conditions (xanthine (XA): 25 µM; xanthine

oxidase (XO): 0.17 U/mL; LPO: 16 nM): without SOD a), and with SOD (205 nM)

b). Both curves were smoothed via adjacent-averaging. B) Cascade reaction in free

conditions (XA: 25 µM; XO: 0.17 U/mL; LPO: 1.6 nM; SOD: 205 nM) for a

gradient of amplex red: a) 12.5 µM; b) 8.4 µM; c) 5.6 µM; d) 3.3 µM and e) 1.7 µM.

Reprinted with permission from Ref. 56. Copyright 2011 Wiley-VCH Verlag GmbH

& Co. KGaA.

In the SOD/LPO cascade reaction, the slope is increased in the

presence of SOD (Fig. 28A b) showing that the reaction completely

detoxifies superoxide radicals via hydrogen peroxide to water and

molecular oxygen. In comparison, when SOD is absent (Fig. 28A a), the

signal intensity is clearly reduced, which was taken into account as

background signal together with amplex red autooxidation. Because

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these effects can be neglected for other activity tests at high enzyme

concentration, the kinetics of encapsulated enzymes (Fig. 28B) will only

be compared to that of free enzymes in solution providing similar

concentration conditions as observed in nanovesicles.

2.3.5. Multi-enzyme kinetics in situ inside polymersomes

Optimisation of the co-encapsulation by the selection of optimal

enzyme concentrations and ratios allows a very efficient cascade

reaction, taking place in situ in the polymersome cavity, where superoxide

radicals were successfully reduced to molecular oxygen and water (Fig.

29A a). Resorufin, the product generated in the step by LPO in presence

of hydrogen peroxide was detected by fluorescent measurements. The

FCS set-up was used in these measurements due to its high sensitivity

and the requirement of only low sample volume.

Fig. 29 A) Cascade reaction of: a) SOD-LPO-loaded nanocontainers with OmpF

inserted inside the polymer membrane; b) SOD-LPO-loaded nanocontainers

without OmpF inserted inside the polymer membrane; c) LPO-containing vesicles

with OmpF inserted inside polymer membrane and d) empty polymer vesicles in the

presence of amplex red. B) Cascade reaction of SOD-LPO-loaded nanocontainers

with OmpF inserted inside the polymer membrane with added amplex red gradient

(XA: 25 µM; XO: 0.17 U/mL): a) 16.7 µM; b) 8.3 µM; c) 4.16 µM; d) 1.7 µM.

Adapted with permission from Ref. 56. Copyright 2011 Wiley-VCH Verlag GmbH

& Co. KGaA.


57

SOD/LPO co-encapsulated polymersomes without OmpF (Fig. 29A b)

showed low resorufin formation, which can be attributed to non-

encapsulated LPO, which is still present in low amount after polymersome

purification and is able to oxidise amplex red in presence of H2O2

generated by xanthine/xanthine oxidase (XA/XO) as a by-product.

Similarly, LPO-encapsulated polymersomes with OmpF, but lacking of

SOD also showed low resorufin formation, which was detected due to the

generation of H2O2 generated by XA/XO (Fig. 29A c). The used triblock

copolymer did not induce amplex red autoxidation (Fig. 29A d). In

contrast, SOD/LPO containing polymersomes with OmpF reconstituted

channels were able to generate a considerable amount of resorufin (Fig.

29A a), which is characteristic for a complete cascade reaction. In direct

comparison to SOD/LPO containing polymersomes without reconstituted

OmpF (Fig. 29A b), resorufin intensity was clearly decreased. These

results are in agreement with FCCS measurements, which already

proved successful co-encapsulation.

SOD/LPO containing polymersomes with reconstituted OmpF could

statistically co-exist with polymersomes without OmpF. Based on the

OmpF concentration employed, approximately 14 OmpF molecules are

embedded in one polymersome. Due to this high amount of channels per

polymersome and the statistical distribution of channels, the probability

that a polymersome contains no channels should be small.

In order to qualitatively analyse this cascade reaction, the Michaelis-

Menten kinetic approach has shown to be a suitable model, e. g. in

characterising HRP in the glucose oxidase/horseradish peroxidase

tandem reaction.138 Even if the signal to noise ratio is at the detection limit

due to being close to the single molecule level, Michaelis-Menten kinetics

provided reliable results.139 Therefore, we applied the Michaelis-Menten

kinetic to extract the kinetic parameters of the SOD/LPO cascade reaction

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in free condition (for single molecules) and inside polymersomes (in bulk:

Fig. 28B and in polymersome condition: Fig. 29B).

Calculation of the Michaelis-Menten constant KM revealed a value of

1.1 ± 0.1 µM in free condition, which is lower compared to the value of

7.3 ± 0.4 µM for the SOD/LPO containing nanoreactors, because the co-

encapsulation is limited by the encapsulation efficiencies of the individual

enzymes as confirmed by FCCS. The individual enzymes containing

polymersomes also contribute to the overall cascade reaction, but this

reaction pathway is less effective than the pathway occurring in the co-

encapsulated polymersomes, because the hydrogen peroxide, converted

by SOD containing polymersomes, needs to exit the polymersome and

need to get in contact with polymersomes containing either SOD/LPO or

only LPO. However, the condition of excess of H2O2 is fulfilled due to the

extremely high SOD reaction speed and the presence of OmpF, which

allows good accessibility of the enzymes so that Michael-Menten kinetics

can be simulated.

With respect to Vmax, a value of 11.1 µMs-1 for enzymes in bulk

compared to the value of 0.02 µMs-1 shows the same trend as for single

enzyme encapsulation, where the conversion rate of the enzyme is higher

in bulk condition, because the substrate needs to pass through the

membrane proteins in order to reach the reaction volume inside the

polymersomes.

2.4. Combating superoxide anion radicals in cells by artificial peroxisomes

Polymersomes are qualified for the use inside cells due to their

intrinsic properties such as high stability and their limited miscibility with

phospholipids.140 Artificial organelles can be designed by the successful

uptake of polymersomes containing the desired enzymes in cells,


59

providing that the enzymatic activity inside the polymersomes is

preserved under cellular conditions. The combination of the naturally

occurring enzymes SOD and LPO in synthetic polymersomes generates a

hybrid system, which shares the functionality and properties of

peroxisomes (morphology and size from 200 nm to 1.1 µm)141 when

implanted in cells.

Fig. 30 A) Schematic view of a cell showing various organelles. B) Peroxisomes are

spherical, cell organelles of sizes in the nanometre range that play a significant role

in the regulation of ROS. C) An artificial peroxisome (AP) is based on the

simultaneous encapsulation of a set of antioxidant enzymes in a polymer vesicle,

with a membrane equipped with channel proteins. The APs serve for ROS

detoxification inside cells. D) Schematic enzymatic cascade reaction occurring inside

the AP and serving to detoxify superoxide radicals and related H2O2. Reprinted


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The aim is to combat reactive oxygen species (ROS) in cells (Fig. 30).

Thus, designing efficient artificial peroxisomes (AP) (Fig. 30C) requires

an understanding of their integration into cells and the influence of their

properties on cellular interactions. Cell integration and preservation of

function needs that toxicity, cell-uptake of the polymersomes, intracellular

trafficking and translocation and functionality in intra-cellular conditions

must be studied.

2.4.1. Integration of artificial peroxisomes in cells

The first requirement of the compatibility to use artificial peroxisomes

(APs) in living systems is to assess possible toxic effect to cells. The

toxicity of APs was evaluated by a MTS assay in the HeLa cell line. APs

exhibit minimal in vitro cytotoxicity in HeLa cells for a period of up to 48

hours (Fig. 31A).


61

Fig. 31 A) Viability in HeLa cells after incubation with different concentrations of

APs (MTS assay). Blue bars: incubation time 24 h, red bars: incubation time 48 h.

B) CLSM images of HeLa cells incubated with APs containing SOD-AlexaFluor-488

and LPO-AlexaFluor-633 for 24 h (Scale bar = 20 µm). C) CLSM images of HeLa

cells incubated with APs containing SOD and LPO-AlexaFluor-633 for different

incubation times. (Scale bar = 20 µm). D) Flow cytometry analysis of APs in HeLa

cells for different incubation times (0-, 2-, 4-, 8-, 18-, 24 h). Reprinted with


This is in agreement with the reported non-toxicity of PMOXA-PDMS-

PMOXA-based copolymers with different block lengths studied in other

cell lines.57,62

The first interaction of an AP with a cell is the interaction with the cell

membrane, which determines a possible uptake in the following step. To

this end, we studied the uptake behaviour into HeLa cells of AP co-

encapsulated with SOD-AlexaFluor-488 and LPO-AlexaFluor-633. By the

use of a dual channel confocal fluorescence microscope, co-localisation

of a fluorescence signal coming from SOD-AlexaFluor-488 and of a signal

from LPO-AlexaFluor-633 showed that both enzymes were uptaken in

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cells (Fig. 31B). This is the first time that two different types of enzymes

capable of working in tandem in polymersomes are uptaken in cells.

Although two different enzymes were loaded in polymer vesicles

earlier by us and others,56,142 here we demonstrate for the first time the

presence of nanoreactors containing both enzymes in cellular system.

Further, we directly evidenced the effect of initial concentration dependent

co-encapsulation efficiencies of SOD-LPO enzymes inside nanoreactors

in intra-cellular conditions (Fig. 32). The concentration of SOD inside

nanoreactors (number of enzymes) can be increased to only a certain

extent, probably due to solubility. This effect was demonstrated by FCS

measurements used to determine the number of SOD per vesicle as a

function of SOD concentration (Fig. 17). However, we observed reduced

SOD fluorescence intensity, due to the interaction of LPO AlexaFluor-633

with the copolymer, which suppresses SOD encapsulation efficiency

during the co-encapsulation process. FCS and FCCS experiments

corroborated well with cellular uptake studies.


63

Fig. 32 Confocal image of HeLa cells incubated with SOD-LPO co-encapsulated in

PMOXA12-PDMS55-PMOXA12 polymersomes prepared with different initial

concentrations. a) Control cells b) initial concentration of SOD (0.25 mg/mL) and

LPO (0.1 mg/mL) c) initial concentration of SOD (0.5 mg/mL) and LPO

(0.2 mg/mL) d) initial concentration of SOD (0.25 mg/mL) and LPO (0.5 mg/mL).


Society.

However, for detailed uptake kinetics experiments, we used APs only

encapsulated with AlexaFluor-633 labelled LPO to simplify the

measurements. Cellular uptake was increased with time from 2h to 18h

indicating time dependent internalisation of APs (Fig. 31C). Fluorescence

intensity varied from cell to cell, suggesting the number of internalised

APs in individual cells is different due to non-specific interactions and

miscellaneous level of direct contact to the cells at the close proximity.

In general, cellular uptake is directly related to the concentration of

nanoparticles. However, aggregation can dramatically influence the dose

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of nanoparticle exposed to a cell, changing the exposed concentration

compared to the initial bulk concentration.143 Aggregation behaviour will

not only affect the concentration of APs exposed to a cell surface, but

also change the properties of the APs by having some enzymes outside

which may mislead to inaccurate activity assessments. To test this

hypothesis, we studied the uptake kinetics of APs containing two different

initial concentrations of LPO-AlexaFluor-633 (0.25 mg/mL and

0.83 mg/mL). As we studied the effect of initial enzyme concentration on

encapsulation efficiency and aggregation (FCS experiments), we carefully

choose those two different initial concentrations. At low concentration

(0.25 mg/mL), no aggregation is observed (Fig. 18A), while the higher

concentrated system (0.83 mg/mL) allows to study aggregation behaviour

that was characterised by FCS (Fig. 19A). The rate of uptake was

quantified by using flow cytometry based on the mean cell fluorescence

corresponding to overall cell population. APs containing LPO-AlexaFluor-

633 (0.25 mg/mL) showed minimal uptake of around 10% after 2 h.

Uptake linearly increased over time and reached a plateau at 85% after

18h: cells were saturated with the uptake of APs after 18h under the used

experimental conditions. This saturation might be due to a depletion of

APs for the uptake or saturation of the storage capacity of cells.144,145 APs

containing LPO-AlexaFluor-633 at a concentration of 0.83 mg/mL forming

aggregates as observed by TEM and FCS showed a dramatic change in

the uptake reaching 60% after 2 h incubation time compared to APs

containing LPO-AlexaFluor-633 (0.25 mg/mL). This indicates that

aggregation can dramatically influence the dose of APs in cellular uptake

as well as the internalisation kinetics.

The uptake kinetics can also depend on the mechanism of the

internalisation pathway.146 The effects of several membrane entry

inhibitors on nanoreactor uptake were examined by first incubating the


65

HeLa cells for 1 h at 37 °C with chloropromazine (10 μg/mL) in order to

inhibit the formation of clathrin vesicles, filipin III (1 μg/mL) to inhibit

caveolae, or amiloride (50 μM) to inhibit macropinocytosis, and then

treating the cells with nanoreactors (100 μg/mL) for an additional 24 h.

Next, the cells were washed twice with PBS (pH 7.2) and analysed with

flow cytometry.

Nanoreactors are generally internalised by cells through various

endocytotic pathways. Understanding the route of entry into cells for

nanoreactors is important because it determines intracellular fate and

therapeutic efficacy. Uptake kinetics can also depend on the mechanism

of the internalisation pathway. Preliminary experiments to detect the

mechanism of internalisation by using specific endocytosis pathway

inhibitors were analysed in flow cytometry (Fig. 33). The quantified data

suggest that at least two different pathways were involved in nanoreactor

uptake; clathrin-mediated endocytosis and macropinocytosis. Cellular

uptake of APs is dramatically hampered by amiloride, an inhibitor of

macropinocytosis (70%). Thus, macropinocytosis is one of the dominant

uptake pathways and is known for non-specific uptake; therefore,

aggregation strongly affects cellular uptake kinetics based on the route of

entry mechanism. Often more than one endocytotic pathway is involved in

the uptake of polymeric nanoparticles and particularly so in the case of

non-specific uptake.147

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Fig. 33 Flow cytometry analysis of LPO encapsulated in PMOXA12-PDMS55-

PMOXA12 vesicles in HeLa cells with different endocytotic pathway inhibitors (red:

non-treated cells, green: cells treated with nanoreactors without inhibitors yellow:

cells treated in advance with nanoreactors and with chlorpromazine, blue: cells

treated in advance with nanoreactors and with dimethyl amiloride, light blue: cells

treated in advance with nanoreactors and with filipin III). Reprinted with


When nanoreactors serve as APs inside cells, endosomal escape is a

critical requirement. To evaluate whether APs escape endosomes or are

trafficked for degradation, we used an endo-lysosome marker, pH-rodo,

and visualised cells by confocal laser-scanning microscopy (CLSM). The

majority of APs were located in intracellular regions, particularly in the

peri-nuclear region, and only few were stayed in endo-lysosome

compartments (Fig. 34A). Based on the fluorescent signals from the peri-

nuclear region, it can be concluded that APs escaped from endosomes.

Co-localisation signals of endo-lysosome compartments and APs imply

that APs were transported through endocytosis. By visualisation of the

intracellular localisation of APs with TEM, further insight into endosomal

escape was gained. TEM micrography proved the presence of structurally


67

intact APs in peri-nuclear regions, with only a few collapsed APs (Fig.

34B). In endosome-like compartments, the APs were present in clearly

reduced number (Fig. 34C). Intact APs escaped from endosomes and

were allocated in the cytoplasm, which is in agreement with the CLSM co-

localisation studies. Escape from endosomes by PMOXA-PDMS-PMOXA

vesicles has already been reported in THP-1 cells.62

Fig. 34 A) CLSM image of HeLa cells incubated for 24 h with APs (blue: nucleus

stained with Hoechst, green: endosome/lysosome stained with pH-rodo, red:

artificial peroxisomes) (Scale bar = 5 µm). B) TEM images of the peri-nuclear

region of HeLa cells incubated with APs for 12 h (Scale bar = 200 nm). C) TEM

images of endosome-like compartments in HeLa cells incubated with APs for 12 h

(Scale bar = 200 nm). D) Protective effect of APs against paraquat by flow

cytometry analysis: viability of untreated cells considered as 100%, (cells), viability

of cells after treatment with paraquat (PQ) and viability of cells pre-treated with

APs for 24 h, and sequentially treated with paraquat for a further 24 h (PQ-APs). E)

Real time ROS detoxification kinetics of APs in: a) cells treated with pyocyanin, and

b) cells pre-treated with APs (8 h) followed by treatment with pyocyanin. Reprinted


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It is noteworthy that similar block copolymer was studied for polymer-

lipid interactions to understand interfacial effects of the tri-block

copolymer with cell membrane. It has been demonstrated that smaller

amounts of the tri-block copolymer system was sufficient to force the lipid

membranes to phase separate and form domains.140 Additionally we have

shown in earlier studies that similar tri-block copolymer based

polymersomes escape the endosomes in THP-1 cells.62 Therefore, it is

clear that APs escape the endosomes possibly due to the ability to force

the endosomal membrane to phase separate thereby causing endosomal

escape.

To assess the APs antioxidant functionality, oxidative injury was

induced by paraquat in HeLa cells, known to induce toxicity involving

production of ROS.148 Since ROS serve as important intracellular

signalling molecules that affect cell viability, this viability in turn is

modulated by the intracellular production of ROS. The viability of the cells

was quantified by staining them with propodium iodide (PI), a fluorescent

and intercalating agent, and subsequent analysis using flow cytometry.

Cells pre-treated with APs provided an around 26% protective effect

against the oxidative stress caused by paraquat, although the number of

enzymes inside APs was low (Fig. 34D). Indeed, the efficient cascade

reaction inside the APs explains that the increased viability corresponds

to protective effect exclusively by APs. In order to determine the

involvement of APs in the ROS detoxification directly in cells, it is

important to be able to monitor ROS generation in intact, living cells. Such

detection is difficult due to the low concentration and high reactivity of

ROS. The short half-life of the ROS requires an immediate and real time

monitoring in the live cells. Live cell analysis of ROS detoxification

kinetics of APs was monitored in real time using highly sensitive ROS

fluorescent probes. We optimised the concentration of pyocyanin to


69

stimulate the ROS in cells without causing cell death and cell suspension

over a period of 30 min, which shows continuous production of ROS.

Stimulated cells treated with APs detoxified the ROS under oxidative

stress in live cells (Fig. 34E). As expected, cell populations demonstrate

significant heterogeneity in rate of ROS generations upon stimulation to

pyocyanin. Thus, individual cells within a population may exhibit different

kinetics of ROS production as well as different detoxification kinetics.

2.5. Conclusion

We engineered artificial peroxisomes by the combination of natural

occurring antioxidant enzymes with synthetic block copolymers, which

form polymersomes, aiding the cellular antioxidant defence mechanism in

case of oxidative stress. Similar to nature, these APs contain SOD and

LPO or CAT, which detoxifies superoxide radicals in situ via hydrogen

peroxide to the harmless products water and molecular oxygen. The

generation of functional nanoreactors, which are able to be cell uptaken

and function as an artificial organelle, represent a milestone in the

development of in vivo cell implants and will open new frontiers in medical

therapy. The main advantage over previous attempts to reduce reactive

oxygen species is that the APs are not limited for example in uncontrolled

release of enzymes. They carry out the function of a cell organelle

permanently. The polymersome properties were optimised by means of

high encapsulation efficiency to allow an enhanced cascade reaction and

to mimic the morphology and function of a natural peroxisome. Enzymatic

activity over time is preserved even longer than in solution, making this

hybrid system not only suitable for therapeutic applications but also for

technical applications such as biosensors. For the therapy of oxidative

stress, we were able to show that the APs escaped in intact form from

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endo-lysosomes and were distributed within the cytoplasm, important for

proper functioning. In addition, viability measurements revealed that the

APs were non-toxic. Concluding the high efficacy of the APs in cells in

detoxification of superoxide radicals, the APs are a significant innovation

in the treatment of cellular ROS and the repair of disturbed cell functions.

This is of particular importance, because disturbed cell functions are

mainly responsible in the development of disease such as Parkinson`s

disease, arterial sclerosis, cancer and many more.


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3. Design of pH-triggered nanoreactors

The design of pH-triggered nanoreactors is based on the

encapsulation of environment-sensitive molecules in polymersomes and

their remote control via specific and functional ion-channels incorporated

in the polymersome membrane. Feasibility of this concept was proven

using lipid vesicles encapsulating pyranine (a pH dependent fluorescent

molecule) and a channel was formed in the lipid membrane with the ion-

channel forming molecule gramicidin A.149 Because lipid vesicles are very

fragile and short-lived, the further goal was to use polymersomes in order

to design stable and pH-triggerable nanoreactors.

Fig. 35 Procedure to prove functional gramicidin channel formation: a) Pyranine

encapsulated vesicles show high fluorescence at pH = 8.3. b) Upon lowering the pH

of the vesicle solution to pH = 5, the fluorescence of non-encapsulated pyranine

drops immediately. c) Upon addition of gramicidin, functional channel formation

lead to a permeabilisation of the vesicles and d) thus to a reduction of fluorescence

of the encapsulated pyranine. e) and f) Pyranine encapsulated vesicles preserve their

fluorescence activity, when no gramicidin was added.

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Specific functions under biological conditions of polymersomes can be

induced by external trigger factors, such as pH, light or temperature. This

is especially useful for therapeutic applications when unwanted side

reactions need to be avoided. A possible strategy to develop responsive

nanoreactors is by combining specific pores, such as gramicidin in the

membrane of polymersomes. Gramicidin incorporation in liposomes was

already reported.150 The proof of functionality of gramicidin in the

membrane was realised by encapsulating a pH-sensitive dye, pyranine, in

the aqueous cavity of liposomes. Upon lowering the pH of the liposome

solution, the quenching of pyranine molecules was directly monitored. In

this case, due to the protective character of the vesicles, encapsulated

pyranine molecules were not quenched and still yielded high fluorescence

intensity. In the next step, upon addition of the gramicidin ion-channels to

the vesicle solution, the pore forming capabilities were proven by direct

observation of the quenching of the dye encapsulated in the vesicles. The

conceptual steps of functional gramicidin incorporation are illustrated in

Fig. 35. After the successful realisation of this technique with liposomes,

the same preparation procedure and execution of the experiments were

performed using polymersomes.

3.2. Evidence of functional ion-channel incorporation in liposomes

Gramicidin appears in different conformations when dissolved in

different solvents151 and not every solvent provides functional pore

formation. First a suitable solvent had to be identified, which allows ideal

pore formation and would not destroy the vesicles. The different solvents

for gramicidin, dimethyl sulphoxide (DMSO), trifluoroethanol (TFE),

dioxane, pyridine, isoporpanol, decane and the mixtures DMSO:ethanol


73

(DMSO:EtOH) in the ratio 1:1 and the mixture TFE:DMSO:EtOH (2:1:1)

were first tested on lipid pyranine-encapsulated vesicles. Fluorescence

spectroscopy results for liposomes were shown in Fig. 36.

Fig. 36 Fluorescence spectrometry: Time driven fluorescence measurement of the

fluorescence emission of pyranine encapsulated in lipid vesicles after lowering pH

from 8.3 to 5 (each measurement around time point of 75 s) and subsequent addition

of gramicidin (in different solvents) after around 160 s.

As expected (due to the pH dependent fluorescence intensity of

pyranine), a decrease in fluorescence intensity occurred after adding HCl

solution. Subsequently, gramicidin in different solvents was added. A

further decrease of fluorescence intensity in liposomes was only

observed, when gramicidin dissolved in DMSO, TFE, pyridine, decane,

DMSO:EtOH (1:1), or isopropanol was added after around 160 s to the

liposome solution, shown in Fig. 36. This indicates that functional ion-

channels were reconstituted in the membrane of the liposomes. In

addition, these used solvents were not able per se to

permeabilise/weaken the liposome membranes, because no change of

signal intensity was observed after the treatment with the solvent.

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3.3. Evidence of functional ion-channel incorporation in polymersomes

The solvents that allowed gramicidin to build a functional pore into the

liposome membrane were then tested with pyranine encapsulated

PMOXA14-PDMS65 polymersomes.


fluorescence emission of pyranine encapsulated in PMOXA14-PDMS65 vesicles after

lowering pH from 8.3 to 5 (after 65 s) and subsequent addition of gramicidin (in

TFE:DMSO:EtOH (2:1:1)) after 110s. Addition of gramicidin (red); addition of

solvent (TFE:DMSO:EtOH (2:1:1)) (black).

HCl was added after 50 s to 80 s and the solvents (with and without

gramicidin) were added after 110 s to 130 s. Only gramicidin dissolved in

TFE:DMSO:EtOH (2:1:1) added to the vesicles lead to a reduction of

fluorescence intensity, without destroying/destabilising the membrane by

the solvent alone (Fig. 37). Note that we observed a permeabilisation of

the membrane, when solely TFE was used. Interestingly, this behaviour

was not observed when liposomes were used.


75

In order to prove structural integrity of the polymersomes after different

steps, preliminary TEM measurements and light scattering experiments

were performed before, after lowering pH and after addition of the

gramicidin dissolved in TFE:DMSO:EtOH (2:1:1). In the first step, TEM

micrographs showed the formation of pyranine-encapsulated

polymersomes in pH 8.3 (Fig. 38A). After lowering the pH of the

polymersome solution from 8.3 to 5, TEM micrography revealed structural

defect vesicles (Fig. 38B).

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Fig. 38 TEM micrographs of A) AB polymersomes containing pyranine. pH inside

and outside of polymersomes = 8.3; B) AB polymersomes containing pyranine.

Image was taken after pH of the solution was reduced to pH = 5; C) AB

polymersomes containing pyranine. Image was taken after pH was reduced to pH =

5 and subsequent addition of gramicidin.


77

Considering that the fluorescence intensity during fluorometric

experiments did not vanish completely when the pH was lowered, the

TEM results suggest that a high proton pressure induced a polymersome

collapse during harsh TEM sample preparation. This explanation is

further supported by TEM micrographs showing intact polymersomes

(Fig. 38C), when the pH of the polymersome solution was decreased,

while gramicidin was incorporated into the polymersome membrane.

Under these conditions, the high proton gradient induced by the pH

change is immediately equalized inside the polymersomes by functional

pore formation.

In addition, gramicidin incorporation also shows no membrane

destabilisation effects. These results further underline preliminary light

scattering experiments (Fig. 39A - C), where the hydrodynamic radii did

not change between the different steps of the experimental procedure; i.

e. A) non-treated polymersomes, B) measurement after lowering pH, C)

measurement after lowering pH and subsequent addition of gramicidin. In

this last step, an additional small peak in the size range of 10 nm

appeared, which can be contributed to the formation of micelles induced

by the solvent of gramicidin.

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Fig. 39 Preliminary dynamic light scattering experiments of A) AB polymersomes

containing pyranine. pH inside and outside of polymersomes = 8.3; B) AB

polymersomes containing pyranine after the pH of the solution was reduced to pH =

5; C) AB polymersomes containing pyranine after the pH was reduced to pH = 5

and subsequent addition of gramicidin.

3.3.1. Ion-channel formation (H+ kinetics) as a function of gramicidin

concentration in polymersome membranes

Different gramicidin concentrations were used to study H+ influx rate

as a function of gramicidin concentration in the membrane of


79

polymersomes. Already at a gramicidin concentration of 65 nM (Fig. 40,

red line), H+ influx can be observed, showing that already a low number of

gramicidin is sufficient for proton trafficking. This is in agreement with the

high monovalent cation conductance of gramicidin.151 A two-fold increase

of the gramicidin concentration to 130 nM showed no significant increase

in the H+ influx rate (Fig. 40, blue line), while at a gramicidin

concentration of 32 nM no pore formation was detected (Fig. 40, black

line).


fluorescence emission of pyranine encapsulated in PMOXA14-PDMS65

polymersomes after lowering pH from 8.3 to 5 (after 125 s) and subsequent addition

of gramicidin (after 300 s) (in TFE:DMSO:EtOH (2:1:1)) at different gramicidin

concentrations (dark line: 32 nM; red line: 65 nM; blue line: 130 nM).

3.4. pH-triggered nanoreactors

Gramicidin incorporation in PMOXA14-PDMS65 polymersomes forms

the basis to engineer pH-triggered nanoreactors, which are composed of

a model pH-sensitive enzyme, acid phosphatase. In order to visualise

enzymatic activity, a suitable substrate need to be co-encapsulated with

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the enzyme. Here the enzyme substrate ELF97 was used, which is

converted into a fluorescent product, upon activation of the enzyme (Fig.

41).

Fig. 41 Schematic view of acid phosphatase and ELF97 co-encapsulated vesicles.

Upon lowering pH to around pH = 5 and subsequent addition of gramicidin in order

to permeabilise the vesicle membrane, the resulting pH condition inside the vesicle

activates acid phosphatase, which produces a fluorescent product from the ELF97

substrate.

In order to prove the concept, acid phosphatase together with ELF97

was co-encapsulated in liposomes. Inside the liposomes, activation of

acid phosphatase by a change of pH to either pH 6 (Fig. 42 blue line) or

pH 5 (Fig. 42 dark line) led to the formation of fluorescent ELF97 product,

when gramicidin formed ion-channels in the vesicle membrane. Under

these conditions, the successful lowering of the pH in the vesicles allowed


81

to activate the enzyme. In contrast, no fluorescent product was formed in

absence of gramicidin (Fig. 42 red line).

Fig. 42 Time dependent fluorescence emission of ELF97 product encapsulated in

acid phosphatase co-encapsulated liposomes: black line) Adjusting pH to around 6

and subsequent addition of gramicidin led to the formation of the fluorescent

ELF97 product; red line) Adjusting pH to around 6 in absence of gramicidin did not

induce product formation; blue line) Adjusting the pH to 5 and subsequent addition

of gramicidin led to an increased formation of the fluorescent ELF97 product.

When the same reaction was performed using vesicles made from

PMOXA14-PDMS65 copolymer, no increase of fluorescence was observed.

Thus, we were not able to generated pH-sensitive polymer nanoreactors.

The reason for this result might be that a low encapsulation efficiency of

the enzyme does not allow for simultaneous co-encapsulation of the

substrate, necessary to detect the enzymatic functionality. To solve this

problem, further optimisation of the individual encapsulation of the

enzyme and substrate is needed in order to allow co-encapsulation.

Another possibility could be the switch to a new enzyme/substrate

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system, which is distinguished with improved properties such as

increased encapsulation behaviour and assay sensitivity.

3.5. Conclusion

The diblock copolymer used here formed vesicles in the diameter of

approximately 200 nanometres and was able to encapsulate a pH

sensitive dye, pyranine. This was used in order to prove the incorporation

of gramicidin in the membrane of polymersomes. This achievement is an

important step towards the design of pH-triggerable nanoreactor, in order

to activate or deactivate the nanoreactor activity upon request via specific

ion-channels. Up to now, encapsulation of a pH sensitive enzyme, acid

phosphatase, together with its substrate is only possible in case of

liposomes generating only a low detection signal. In case of

polymersomes, no signal could be generated, indicating no simultaneous

co-encapsulation of the enzyme and its substrate. Further investigation to

improve encapsulation efficiency of the enzyme and the substrate to

achieve pH-sensitive nanoreactor are on-going. It is also considered to

exchange the enzymatic system in order to improve the system’s

performance and thus to engineer functional pH-switchable nanoreactors.


83

4. Metal-functionalised polymersomes for molecular recognition

The specificity exhibited by molecular recognition assemblies is based

on a perfect structural match between biological entities, which results in

minimisation of energy, allowing the energy barrier to be by-passed and

to fulfil a biological role. The fact that molecular recognition is based on

highly selective moieties, their application is discussed in industrial and

medical context, such as the purification102,103 and immobilisation21,23,104,105

of biomolecules, labelling of proteins,106,107 2D-crystallisation23,104 and

biosensing.152,153 Molecular recognition also plays an important role in the

development of nanocarriers for drug/protein delivery or nanoreactors due

to the potential decrease of dosages and improved localisation in a

desired biological compartment. In this respect, these carriers need to be

functionalised with a specific affinity tag to enable molecular recognition.


In a molecular recognition process, solvent molecules are specifically

replaced by ligands. During this process, a change of geometry can occur

at the binding site, which allows specific recognition. Even though the

principle is widely applied in technology, the change of geometry and the

exact mechanism are in many cases poorly understood.

Here, we characterise in detail the conformation change of the

coordination sphere of CuII-trisNTA centres, which were used to

functionalise poly(butadiene)-b-poly(ethylene oxide) (PB-b-PEO) diblock

copolymers. These metal-functionalised block-copolymers are able to

form polymersomes in aqueous solution with metal centres exposed at

the inner and outer surface (Fig. 43A), while only the outer exposed

metal centres were used for binding studies. We used trisNTA (Fig. 43E)

for functionalisation, because its binding affinity is higher than NTA (Fig.

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43C). His6-tags with optional labelling can be used to bind to these

molecular architectures (Fig. 43D). The use of His6-tags is regarded to

be a simplified model of His6-proteins, which were used to bind to metal

sites of the polymersomes` surface. This simplification of using short His6-

tags allows better understanding of the docking mechanism, which is

essential for biological, technical and medical applications. Compared to

Ni2+ ions, which are used in many NTA-based systems that have an

octahedral coordination sphere, different coordination geometries exists

for Cu(II).154 The different arrangements of Cu(II) are induced by its

stereochemistry and allows us to use copper as the metal-coordination for

His6-tags and to gain a deep insight into the rearrangement of the metal

centre upon binding of a tagged molecule.

The high affinity of different His-tagged proteins to metal-NTA

functionalised liposomes and PB-b-PEO diblock copolymer

polymersomes is widely known.21,23,104 Because the dissociation constant

of His-tagged proteins is more or less independent of the surface where

the metal centre is bound, the local environment of the metal is crucial

during the binding process.


85

Fig. 43 A) Schematic representation of CuII

-functionalised PB-b-PEO vesicles. The

metal centres available for binding are located on the outer vesicle surface that can

be recognised by fluorescently labelled His6-tags; B) Detailed view of A); C)

Schematic formula of CuII

-NTA complex and D) fluorescently labelled

(sulphorhodamine B-His6-tags; E) CuII

-trisNTA. X represents solvent molecules or

external binding atoms of other molecules. The red star represents

sulphorhodamine B. Reprinted with permission from Ref. 55. Copyright 2012


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4.2. Formation of Cu(II)-trisNTA functionalised polymersomes

Using the diblock copolymer PB39-PEO36-SA-OH-containing 10% PB39-

PEO36-SA-trisNTA.d-Cu2+ and applying the film rehydration method led to

the self-assembly of polymersomes. In order to reduce the polydispersity

of polymersome sizes, ranging from giant vesicles to nanovesicles, the

sample solutions were extruded through a 1 µm, 400 nm and 200 nm

pore-sized filter membrane in descending order. The use of copper

instead of nickel has the advantage of studying the copper

stereochemistry and to determine the final structural changes in the

metal-coordination environment by EPR while a high binding affinity is

provided by Cu(II).

Fig. 44 A) Dynamic light scattering representation. Data extrapolated to zero

concentration and zero angle of vesicles made from 10% CuII

-functionalised PB39-

PEO36 block copolymer; B) Guinier plot representation of static light scattering

data of vesicles made from 10% CuII

-functionalised PB39-PEO36 block copolymer.

The data is extrapolated to zero concentration and zero angle. Reprinted with


Dynamic light scattering155 was used to characterise the polymersome

hydrodynamic radius to a value of Rh = 107 ± 12 nm (Fig. 44A),

comparable to NiII-functionalised trisNTA-PB-b-PEO vesicles.23 Static light

scattering experiments (Fig. 44B) allow to calculate the radius of gyration

(RG) and the ratio of RG/Rh was determined subsequently as 0.96, which

indicates a hollow-sphere morphology.155


87

As expected, size and morphology of the polymersomes were not

affected by using similar copper concentration compared to nickel inside

the NTA moieties.23 This also indicates that the metal ions are complexed

in the NTA pockets of the trisNTA and do not affect the polymer

membrane. Further, static light scattering characterisation provided the

weight average molecular weight of a vesicle (Mw) to be 8 x 107 ± 3 x 106

gmol-1 and the second virial coefficient A2 to be close to zero, ruling out

any long-range interactions between the vesicles in the investigated

concentration range. In addition, the polymersomes preserved their size

and shape over several months at room temperature.

Another characteristic of polymersome formation is the aggregation

number, which is obtained by dividing the weight average molecular

weight of a vesicle by the weight average molecular weight of the diblock

copolymer. The value calculated for this system is 20,000, which is typical

for nanometre-sized polymersomes.24

This aggregation number can be used to qualitatively analyse the

number of functional metal ions on the outer polymersome surface. There

is a 50% probability that the used Cu-trisNTA-functionalised diblock

copolymers protrude at the outer surface. Further, it has to be considered

that only 10% of polymers used were functionalised, which, in addition,

decreases the percentage of active available functional groups.

Therefore, based on the assumption that only 10% of block copolymers

was rehydrated during the film hydration process, we estimated that a

polymersome contains approximately 300 metal ions, which corresponds

to 20 copper ions per 100 nm2 of polymersome surface (nanometre-sized

polymersomes).

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4.3. His6-tags binding to CuII-trisNTA polymersomes

When His6-tags bound to the nanometre-sized CuII-trisNTA

polymersomes, no change in morphology or structure was observed,

demonstrating the high stability after the recognition process (Fig. 45).

Also, no aggregate formation was induced upon binding of the tag-

molecules, which could affect cellular-uptake behaviour for potential

therapeutic applications. This was shown by TEM micrographs of 10%

copper-functionalised polymersomes, before and after the addition of

His6-tags (Fig. 45A and B). In both cases, the size of the polymersomes

is consistent with the values measured with light scattering. Contrast

differences in the TEM micrographs of non-bound Cu-trisNTA-

functionalised polymersomes compared to the same polymersomes with

attached His6-tags are likely due to different capillary effects.

Fig. 45 TEM micrograph of 10% copper-functionalised polymersomes. A) CuII

-

trisNTA-functionalised PB-b-PEO polymersomes; B) His6-tag bound to CuII

-

trisNTA-functionalised PB-b-PEO polymersomes. Reprinted with permission from

Ref. 55. Copyright 2012 American Chemical Society.


89

To visualise His-tag binding with confocal laser-scanning microscopy,

we generated giant CuII-trisNTA-functionalised PB-b-PEO polymersomes

and incubated them with fluorescently labelled-His6-tags (s-His6-tags). To

reduce the background fluorescence intensity of the images, non-bound

peptide (in a small amount) was removed by dialysis. In the laser-

scanning micrographs, polymersomes specifically bound with s-His6-tags

appear as bright fluorescent coronas (Fig. 46A). Because the PB-b-PEO

polymersome membrane is highly impermeable to small molecules, the

fluorescent s-His6-tags were not able to penetrate the membrane. The

hollow vesicular structure of the peptide bound polymersomes could be

clearly rendered by the composition of fluorescent z-stack images taken

in different layers of the sample (Fig. 46B).

Fig. 46 Confocal fluorescence microscopy image of fluorescently labelled His6-tags

bound to the outer surface of PB39-PEO36-SA-/ 10%-PB39-PEO36-SA-trisNTA.d-Cu2+

(800 µM) vesicles incubated with s-His6-tags in bidistilled water. A) Two-

dimensional cut, focused on the mid-plane of the selected vesicles; B) three-

dimensional image produced by merging two-dimensional layers measured at

different heights. Reprinted with permission from Ref. 55. Copyright 2012


Fluorescence correlation spectroscopy (FCS) is widely used to

investigate molecular interactions of fluorescent molecules, such as green

fluorescent protein interaction to an antibody.156 Here, this technique is

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used to study the binding of fluorescently labelled His6-tags to nanometre-

sized polymersomes, based on the change of diffusion times, which is

different from small molecules (for example fluorescently labelled His6-

tags) compared to big entities, for example nanovesicles. CuII-

functionalised polymersomes (at a concentration of 8 µM) incubated with

s-His6-tags (initial concentration of 50 nM) showed two populations. A

small portion (< 5%) of a population with a diffusion time of 56 µs

represents free peptides still present in solution. The other population, to

a portion of more than 95%, has a characteristic diffusion time of 7.5 ms,

which can be attributed to s-His6-tags bound to vesicles (Fig. 47A).


91

Fig. 47 A) FCS autocorrelation curves and the simulation of a) sulphorhodamine B

acid chloride labelled His6-tags; b) sulphorhodamine B acid chloride labelled His6-

tags bound to vesicles; B) Fraction of bound s-His6-tags as a function of the CuII

content of PB39-PEO36-SA-OH/ 10%PB39-PEO36-SA-trisNTA-Cu2+

vesicles

generated in bidistilled water. (Initial concentration of His6-tag: 50 nM, and initial

concentration of Cu-trisNTA functionalised vesicles: 8 µM). Reprinted with


Similar experiments of His6-red fluorescent protein to NiII-

functionalised PB-b-PEO polymersomes exhibited a similar binding

behaviour, proving that our His6-tag model system can be used to check

binding properties of much larger His-tagged proteins. The small size of

the model peptides should also cause less interference with the vesicle

surface, which in turn allows faster and stronger binding behaviour.

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FCS results are in agreement with the observations from confocal

microscopy, where the majority of s-His6-tags are bound to the metal-

functionalised polymersomes (Fig. 47A and B). From the diffusion time

obtained by FCS, the hydrodynamic radius Rh can be determined via the

Stokes-Einstein equation to be 90 nm − comparable to both values

obtained from light scattering and TEM.

The dissociation constant of s-His6-tags bound to Cu2+-functionalised

polymersomes was determined by a titration of the s-His6-tags (50 nM)

with increasing amounts of polymersomes (Fig. 47B). The Cu2+ of the

vesicle was varied from 2 to 8 M. The corresponding curve was fitted by

a Langmuir isotherm21,157 (equation 7) and the binding constant was

determined.

(7)

F corresponds to the corrected fraction of His6-tags-bound vesicles

and P is the metal concentration of the related samples. K stands for the

binding constant. A dissociation constant KD of s-His6-tags was calculated

to a value of 0.6 ± 0.2 µM. In comparison to other KD of similar systems

shown in Table 1, this value is lower, which stands for increased binding.

Table 1 Different KD-values in polymersomes and liposomes using different His-

tagged ligands.

Vesicle system Ligand KD [µM] (FCS-

based) Ref.

PB60-b-PEO34-SA/PB60-b-PEO34-SA-NTA.d-Ni2+ His6-EGFP 12.0 ± 0.8 21

PB39-b-PEO36-SA/PB39-b-PEO36-SA-trisNTA.d-Ni2+ His6-RFP 2.0 ± 0.4 23

PB39-b-PEO36-SA/PB39-b-PEO36-SA-trisNTA.d-Ni2+ His10-MBP-

FITC 7.0 ± 1.2 21

PB39-b-PEO36-SA/PB39-b-PEO36-SA-trisNTA.d-Ni2+ His6-EGFP 12.3 ± 1.2 21

Ni2+-NTA-NBD-lipid His6-peptide 4.3 ± 0.8 157

PB39-b-PEO36-SA/PB39-b-PEO36-SA-trisNTA.d-Cu2+ His6-tag 0.6 ± 0.2 this work


93

In Table 1, nickel-functionalised vesicles were used for protein binding

for comparison with our copper-functionalised systems. It was shown by

molecular dynamics simulations that NiII and CuII have a similar affinity

towards a His6-tag.120 Nevertheless it was shown that copper binds His-

tags more strongly than nickel, providing a high binding capacity, but a

poorer specificity.

Comparing the His6-tags binding of our copper-functionalised

polymersomes with His6-RFP binding to PB39-PEO36-trisNTA.d-Ni2+

vesicles, a much greater binding affinity can be stated. We assume that in

addition to the different binding affinity as explained before, the small

peptide molecules interfere less with the flexible PEO chains at the

polymersome surface, so that improved binding conditions were provided.

Polymersomes containing no metal-functionalised block copolymers

showed less than 3% His6-tag binding measured under the same

conditions. This low unspecific binding of the His6-tag is in agreement with

the protein repellent character of PEO chains at the vesicle surface.22

Also non-functionalised vesicles did not bound s-His-tags after addition of

Cu(OTf)2, clearly defining that copper complexed in the NTA pocket is the

recognition point of His-tag proteins.

FCS can be also used to quantify the number of bound s-His6-tag per

polymersome by performing brightness measurements. The molecular

brightness of peptide-bound polymersomes recorded as counts per

molecule (CPM) must be divided by the CPM of freely diffusing s-His6-

tags, taking the fluorescence quantum yield into account. This calculation

resulted on average in 210 ± 36 molecules per polymersome. Compared

to the value of reported His6-RFP binding to vesicles, s-His6-tags bound

an order of magnitude higher to the polymersomes of similar nature.

P. Tanner

94

When the small size of the used labelled His6-tags compared to the size

of a fluorescent protein is considered, we can hypothesise that more

metal-trisNTA moieties can be more easily occupied with small peptides,

which explains the small KD value (Fig. 43A and B). Furthermore, the

calculated number of His6-tags per polymersomes is in agreement with

the available metal ions/polymersomes calculated from the aggregation

number determined by light scattering.

4.4. Structure of the coordination sphere of CuII in His6-tag-Cu

II-trisNTA

polymersomes in comparison to model systems

In order to gain more insight inside the coordination sphere of Cu(II)

before and after the binding process of the His-tag molecules, electron

paramagnetic resonance spectroscopy (EPR) was applied. EPR can be

used to characterise structural information of paramagnetic species, e. g.

transition metals or radicals. The observed X-band CW-EPR spectrum of

polymersomes (Fig. 48a) produces only a broad signal with a low

intensity, which can be ascribed to a high local copper concentration

coming from the high number of metal ions per polymersome as

calculated from light scattering data. In this case, the resulting strong

dipolar interactions of the CuII centres are potentially responsible of

broadening of the EPR spectrum to the detection limit. On the other hand,

EPR measurements of CuII-functionalised PB39-PEO36-SA-trisNTA.d.21

provided clear spectra. The CW-EPR spectrum of the functionalised

polymersomes (Fig. 48a) is different from the simulated spectrum, where

the reported EPR parameters of the latter building blocks were used for

the polymersomes (Fig. 48b). Because the spectrum in polymersomes

(Fig. 48a) exhibits spectral features, which were characteristic of strong

dipolar interactions between CuII sites, it cannot be simulated using a

single species.


95

Fig. 48 (a) X-band CW-EPR spectrum of a frozen solution of CuII

-trisNTA

functionalised vesicles in bidistilled water taken at 100 K. (b) Simulation of the CW-

EPR spectrum of the PB39-PEO36-SA-trisNTA.d using the parameters reported in

Ref. 20

. (c) Experimental X-band CW-EPR spectrum of a frozen solution of CuII

-

trisNTA functionalised vesicles incubated with the His6-peptide in a 1:50 ratio taken

at 10 K. (d) Simulation of (c) using the parameters in Table 1. (e) and (f) individual

contributions to the simulation (d). All presented spectra are normalised for clarity.

The spectral intensity of (a) is more than a factor 10 lower than that of (c).


Society.

When we incubated the polymersomes with His6-RFP, His6-EGFP or

with His6-tags, only the addition of His6-tags, at an initial concentration of

2 mM, led to a change in the EPR spectra compared to the non-bound

condition, observable in an increased CW-EPR signal. This behaviour is

caused by the lower concentration of His6-proteins used, which is limited

by their commercial availability and the function of the peptide to act as a

spacer between the different CuII sites. In Fig. 48c, the X-band CW-EPR

spectrum together with its simulation (Fig. 48d) of His6-peptide incubated

polymersomes is shown. Two contributions can be discerned (EPR

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96

parameters are given in Table 2), although the signal is poorly resolved.

The EPR parameters are typical of type II copper centres.158 The

vesicle:His6 system is in agreement with the situation where CuII is

equatorially ligated either to four oxygen atoms (4O) or to one nitrogen

and three oxygen atoms (1N3O) as the first EPR parameters. The second

types imply that either two nitrogen atoms and two oxygen atoms (2N2O),

three nitrogen atoms and one oxygen atom (3N1O), or four nitrogen

atoms (4N) are coordinated with CuII, whereas the last set supports

histidine binding. In this case, high values for the z-component of the

copper hyperfine have been reported.159-163 The third contribution caused

by the copper sites located on the inner vesicle surface will be present at

considerably lower intensity, which is in agreement with the spectrum in

Fig. 48a.

Different model systems have been studied to interpret the above

findings: a) a mixture of Cu-triflate (Cu(OTf)2) with NTA, b) a mixture of

Cu(OTf)2 with His6-peptide and c) a mixture of Cu(OTf)2 with His6-tags

and NTA. The obtained EPR parameters are presented in Table 2. It is

evident that the His6-tags cannot bind to free CuII ions under the given

conditions. This possibility was excluded by the fact that the spectral

changes did not occur (Fig. 48), when peptide was added to non-

functionalised vesicles containing free CuII ions in the solution. Moreover,

EPR experiments made with different mixtures of Cu(OTf)2, NTA and the

His6-peptide showed that both NTA and the peptide ligate to the Cu(II)

ion. Also, it became obvious that more CuII centres are linked with

increasing His6-tag concentration.


97

Table 2 Principal g and copper hyperfine values of CuII

-trisNTA functionalised

polymersomes compared with different model systems. The hyperfine values are

given for the 63

Cu nucleus.

gx, gy gz |Ax|,|Ay|/MHz |Az|/MHz

CuII vesicles with His6 2.035 (± 0.004)

2.066 (± 0.005)

2.230 (± 0.001)

2.215 (± 0.001)

5 (± 20)

5 (± 20)

570 (± 1)

590 (± 2)

Cu(OTf)2:NTA (1:5) 2.061 (± 0.005) 2.302 (± 0.001) 45 (± 20) 512 (± 2)

Cu(OTF)2:His6 (1:5) 2.074 (± 0.005) 2.375 (± 0.001) 21 (± 20) 450 (± 2)

Cu(OTf)2:NTA:His6 (1:5:5) 2.058 (± 0.005) 2.282 (± 0.001) 10 (± 20) 510 (± 2)

PB39PEO36-SA-trisNTA.d 2.052 2.253 91 517

Cu(His) solution at pH 7.3

(Ref. 162) 2.058 2.24 37 561

Cu(His) solution at pH 3.8

(Ref. 159) 2.06 2.31 - 507

A hexacoordination of the metal site is typically assumed (Fig. 43C)

for aqueous transition-metal complexes of NTA, which involves the

ligation of three carboxyl groups, an amine group and two water

molecules. Although no structural data are available, a similar binding

mode is proposed for the interaction between a polyhistidine tag and

metal-NTA chelates. The crystal structure of a bisimidazole complexed to

CuII and NTA shows a five-coordinate square pyramidal structure, with

one protonated carboxyl group, two imidazoles and two carboxy groups of

NTA equatorially ligated, and a weak axial coordination of the NTA amine

group.23 Therefore pulsed EPR experiments were performed to verify this

structure. The low-frequency area of the HYSCORE spectrum of a

Cu(OTf)2:NTA (1:5) mixture exhibits no cross peaks from a weakly

coupled 14N nucleus as expected when amine nitrogen atoms binds

axially to copper. The Mims ENDOR spectra (Fig. 49) and the 1H

HYSCORE spectra, taken at different magnetic field positions, show

maximum 1H hyperfine couplings of 7 to 9 MHz.

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98

Fig. 49 Mims-ENDOR of Cu(OTf)2:NTA mixture of 1:5 ratio in frozen water

solution recorded (a) at B0 = 344.8 mT (observer position corresponding to g ≈ gx,y),

and (b) at B0 = 303.0 mT (observer position corresponding to g = gz, MI = -1/2).


Society.

This is in accordance with the findings for [Cu(H2O)6]2+ in frozen

water164 and in Mg(NH4)2(SO2)4.6H2O crystals165 and corroborate with the

equatorial water ligation in the CuII-NTA complex. The large proton

coupling is not likely to be caused by the NTA protons, as their far

distance from the copper site would result in smaller hyperfine couplings.

Davies ENDOR spectroscopy was used to probe the interactions with

directly coordinated nitrogen atoms. A value of ~25 MHz for the z-

component of the hyperfine interaction of the amine nitrogen was

obtained, confirming an equatorial ligation of this amine.

A low-frequency HYSCORE spectrum was obtained by adding His6-

tags to a Cu(OTf)2:NTA mixture, which shows contributions of a weakly

coupled 14N nucleus (Fig. 50A) simulated by using the data in Table 3

(Fig. 50B). The obtained parameters resemble the reported values for

the remote imidazole nitrogen in CuII-bound imidazole complexes166 and

CuII-bound histidine complexes.159,161,167,168 This underlines the His6-


99

peptides binding, seen in CW-EPR measurements. Furthermore, the

Mims ENDOR and HYSCORE spectra indicate a maximum proton

coupling of 5.8 MHz (Fig. 51). This corresponds to the known proton

hyperfine coupling value for the Hε proton of an equatorially ligated

histidine imidazole to be max. 5.4 MHz.162 Therefore, the water,

equatorially ligated to copper centre, is substituted with His residues.

Fig. 50 A) X-band HYSCORE spectrum of a frozen solution of

Cu(OTf)2:NTA:His6-peptide mixture at a 1:5:50 ratio taken at 5 K; B) Simulation of

A) using the parameters from Table 3. The observer position is 335.0 mT, τ = 104

ns. Reprinted with permission from Ref. 55. Copyright 2012 American Chemical

Society.

P. Tanner

100

Table 3 Comparison between the experimental 14

N spin Hamiltonian parameters

derived from the HYSCORE spectra for His6-tag ligation and the CuII

-NTA

functionalised vesicles and the CuII

-NTA complexes.

A/MHz e2qQ/h/MHz h

CuII vesicles with

His6 ± 1.9 (± 0.3) ± 1.5 (± 0.1) 0.95 (± 0.10)

Cu(OTf)2:NTA:His6

(1:5:50) ± 1.7 (± 0.3) ± 1.5 (± 0.1) 0.90 (± 0.10)

Cu(His) (Ref. 107) 1.4 -1.43 0.95

The pulsed EPR analysis described here allowed to assess the effect

of His6-tag addition to CuII-NTA functionalised polymersomes. Only a

weak spin echo signal of a frozen solution containing the polymersomes

without peptide was obtained and no pulsed EPR experiments could be

performed in agreement with the CW-EPR spectrum (Fig. 48a).

Fig. 51 1H Mims-ENDOR spectrum of a frozen solution of Cu(OTf)2 with NTA and

His6-peptide mixture taken at 5 K at the field position B0 = 334.0 mT, τ = 120 ns. A

maximum 1H hyperfine coupling of 5.8 MHz is observed. Reprinted with permission

from Ref. 55. Copyright 2012 American Chemical Society.

After the addition of the His6-peptide, the echo intensity improved,

which could be explained by the peptide acting as a spacer between the

CuII sites on the polymersome surface, thereby reducing the dipolar


101

interaction between these sites. Nevertheless, a low echo intensity was

still observed so that ESEEM or ENDOR experiments could only be

performed at the position of highest echo intensity (corresponding to the

observer position g = gx,y). This is shown in the 14N HYSCORE spectrum

(Fig. 52A) with its corresponding simulation (Fig. 52B) using the given

data in Table 3. The data confirms a binding of the His6-tags to the copper

site, as the parameters match with data, which is expected for the remote

nitrogen of a His ligand. In addition, Mims-ENDOR experiments arrived to

the same conclusion and are against an equatorial H2O ligation.

Fig. 52 A) X-band HYSCORE spectrum of a frozen solution of CuII

-NTA-

functionalised vesicles with His6-peptide at a 1:50 ratio taken at 5 K; B) Simulation

of a) using the parameters from Table 3. The observer position is 338.8 mT, τ = 120

ns. Reprinted with permission from Ref. 55. Copyright 2012 American Chemical

Society.

Combining all data, EPR analysis of the aqueous CuII-NTA- and the

CuII-NTA-His6 complexes clearly shows the existence of type-II copper

complexes. Pulsed EPR and ENDOR data clarified that the copper ion is

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102

equatorially bound to at least one water molecule and to the NTA amine

nitrogen. This is different from the observation of the X-ray structure of

copper(II)-nitrilotriacetic acid-bis(N-methylimidazol-2-yl)ketone ternary

complexes where the NTA amine is in an axial position.169 In this case, it

is reasonable that a flat bis-imidazole complex may be the cause of this

different configuration.

Upon addition of His6-tags to copper-trisNTA functionalised

polymersomes, the equatorial water is replaced by His residues, as

clearly shown by HYSCORE and ENDOR data. This is further

corroborated by pulsed EPR data, which confirms an equatorial ligation of

the imidazole(s) of His6-tags attached to the CuII-NTA moieties on the

surface of polymeric vesicles.

Incubation of His6-tags to the polymersomes provoked the formation of

two different type-II copper sites (Table 2) as established from CW-EPR.

Their principal g and copper hyperfine values significantly differ from

those observed for the Cu(OTf)2:NTA:His6-peptide mixture. This could

suggest that the surrounding structure influenced the local copper

complex structure. Hence, the gz value of CuII-PB39-PEO36-SA-trisNTA.d

is also lower than that observed for the aqueous copper NTA complex.

The EPR parameters of the vesicle/His6-tag mixture resemble the values

obtained for copper histidine complexes at neutral pH,162 while the

parameters of the Cu(OTf)2:NTA:His6-peptide mixture were comparable to

those of the copper-histidine complexes at pH 3.82.102 The copper-

histidine ligation difference at different pH values originates form the

number of His residues and corresponding amine groups ligating to the

copper centre.

DFT computations complemented these observations. While it is

known that DFT cannot exactly reproduce EPR parameters of transition-

metal complexes in means of underestimated g values and dissatisfying


103

transition-metal hyperfine values yet, the computations can be still used

to estimate general trends and hyperfine values of the ligand nuclei.170

Therefore, the EPR parameters of three models were computed using

DFT (Fig. 53A, B and C). The effect of the exchange of water by His

residues was simulated and the impact of the overall charge of the

complex on the EPR parameters was evaluated. In model A, one NTA

and two water molecules are ligated to the copper site, while models B

and C includes the ligation of one NTA and a bis-imidazole molecule with

a peptide-like linker. While in model C all carboxy groups are

deprotonated, models A and B have one protonated carboxy group. All

structures were optimised in terms of their overall lowest energy state via

geometry optimisation using different starting geometries. The

corresponding coordinates of the models are given in the Appendix. The

DFT results are presented in Table 4 and the corresponding spin density

distributions (Fig. 54 – Fig. 56) are shown in the Appendix. The computed

parameters of models A and B reproduce different experimentally

observed trends: Both typically have a quasi-axial g tensor, characteristic

of type-II complexes with the copper in a dx2-y2 ground state. The

computations confirm for model A, the aqueous CuII-NTA model, the

experimental observation of the NTA amine nitrogen located in the

equatorial plane (i.e. plane determined by dx2-y2) located NTA amine

nitrogen and bearing a considerable amount of spin density. The

computed z-component of this hyperfine interaction (27 MHz) resembles

to the experimental value (25 MHz). Also, the computed maximum

hyperfine interactions (absolute value) of equatorially ligated water

protons (10.7 MHz) is in the same order of magnitude as the experimental

values (9 MHz).

P. Tanner

104

Fig. 53 DFT-optimised structures of the three models. Model A:

[Cu(N(CH2COO)2(CH2COOH))(H2O)2]; Model B:

[Cu(N(CH2COO)2(CH2COOH))(C11H15N5O)]; Model C:

[Cu(N(CH2COO)3)(C11H15N5O)]-. Reprinted with permission from Ref. 55.

Copyright 2012 American Chemical Society.

Following the experimental trend of computed maximum proton

hyperfine couplings, the value for model B (His residues) are smaller (6.4

MHz). Indeed, the hyperfine values of the remote nitrogen atoms are

around 1.5 – 2.2 MHz. In agreement to the experimental results, gz

decreases upon attachment of the His residues and the copper |Az|

values are similar for both models A and B. To conclude, these results

agree with the experimental trends and confirm the EPR parameters,

which indicates that addition of His6-tags to Cu(OTf)2:NTA is entirely due

to the replacement of the equatorial water by His residues.

In a next step, protonation effects of the NTA carboxyl groups are

studied by comparing model B (one protonated carboxyl group) and

model C (no protonation of the carboxyl groups). Deprotonation of the

third carboxyl group caused an increase of the gz value and the g tensor

to become rhombic. The copper |Az| value and the NTA amine nitrogen

hyperfine coupling are reduced considerably, while the rest of the ligand

hyperfine values are less affected ranging in the order of the experimental

errors. Thus protonation could play a role in the different EPR parameters

of the Cu(OTf)2-NTA-His6 mixture and the polymersome-His6 systems.


105

Similar hyperfine values were found in both systems for the protons and

the remote nitrogens of the His residues, which were also computed for

both models B and C. Lower gz (higher |Az| values) were seen for the

polymersome system compared to the Cu(OTf)2-NTA-His6 mixture.

Additionally, the copper |Az| values differ more between the two His6-

ligated systems than between Cu(OTf)2-NTA and Cu(OTf)2-NTA-His6. A

similar trend is also observed for a higher copper |Az| value difference

between models B and C compared to models A and B. These results

suggest that the NTA carboxy groups on the polymersome system are

protonated, probably induced by polymersome surface properties. The

presence of two different copper complexes in the polymersome system

could result from changes in the number or orientation of the His ligands.

P. Tanner

106

Table 4 Computed principal g and hyperfine values for models A-C (Fig. 53).

gx gy gz

Model A 2.067 2.075 2.210

Model B 2.059 2.061 2.193

Model C 2.038 2.099 2.202

nucleus Ax [MHz] Ay [MHz] Az [MHz]

Model A Cu

N(NTA)

H (H2O)a

69

27

-10.8

77

45

6.2

-522

27

-5.4

Model B Cu

N(NTA)

N1(His)b

N2(His)c

H (His)a

49

27

44

1.7

6.4

76

41

36

2.3

-0.4

-533

27

35

1.6

0.1

Model C Cu

N(NTA)

N1(His)b

N2(His)c

H (His)a

173

20

42

1.6

5.9

-3

33

34

2.2

-0.3

-495

20

33

1.6

0.2

aData for the proton with the largest hyperfine coupling (in absolute value), bAverage of

computed hyperfine values of copper bound His nitrogens; cAverage of computed hyperfine

values for remote His nitrogen atoms.

4.5. Conclusion

The CuII-trisNTA-functionalised PB-b-PEO polymersomes are well

suited to study molecular recognition process of peptide binding. As

peptides, we used a model system of an oligohistidine sequence

consisting of six histidines – a His6-tag – to study their binding to copper

centres on the polymersome surface. Upon binding, a detailed structural

view of the rearrangement of the metal coordination sphere was obtained.

Qualitative binding analysis revealed that more than 200 fluorescently

labelled His6-tags were bound to one polymersome. This was visualised

by fluorescence laser-scanning microscopy as a highly intense

fluorescent corona surrounding the metal-functionalised polymersomes.

FCS measurements revealed that this system has a high binding affinity,

which was found to be higher than of similar systems using His-tagged


107

proteins.21,23 Different EPR and ENDOR methods were used to

investigate the electronic and geometric change of the copper centre

complexed in the NTA pocket upon binding of the model His6-tags. His6-

tag binding was demonstrated via the typical HYSCORE signature of the

remote nitrogen of the imidazole ligand of the residue, and by the

reduction of the proton hyperfine coupling using EPR and DFT data from

model systems. As expected from other experiments, these findings

confirmed the strong and selective binding. The highly specific binding

capacity of the copper-functionalised PB-b-PEO polymersomes together

with a simple preparation procedure and inherent high structural stability

make the system very suited for a wide range of applications such as

protein crystallisation, targeting approaches or immobilisation on solid

supports.

P. Tanner

108


109

5. Overall conclusion

We designed and developed protein-polymer assemblies to engineer

artificial organelles. The development required to study three key aspects

in order to increase the overall efficacy of the artificial organelles: i)

increased efficacy by means of optimised building blocks, ii) introduction

of specific membrane permeability by the use of selective ion-channels

and iii) applying specific recognition points by the use of a molecular

recognition strategy:

i) Combination of natural occurring antioxidant enzymes with synthetic

block copolymers, which are able to self-assemble into polymersomes,

allowed engineering of artificial peroxisomes (APs). They can support

natural peroxisomes and help the cellular antioxidant defence mechanism

in general to combat oxidative stress. As active compounds, SOD and

LPO or CAT were used in complex, optimised conditions, which detoxify

in tandem superoxide radicals in situ via hydrogen peroxide to harmless

products. These APs were able to be taken up by cells and function as

specific artificial organelles, opening a new direction in medical therapy.

The polymersome properties were optimised by means of high

encapsulation efficiency in order to allow enhanced cascade reactions

and to mimic the morphology and functions of natural peroxisomes.

Enzymatic activity is preserved over time even longer than in solution,

making this hybrid system not only suitable for therapeutic applications,

but also for technical applications such as biosensors. For the therapy of

oxidative stress, we were able to show that the APs escaped intact from

endo-lysosomes and were distributed within the cytoplasm, which is

important for properly functioning. In addition, viability measurements

revealed that the APs were non-toxic.

ii) In order to address the polymersome membrane permeability in

more detail, in addition to the incorporation of OmpF in triblock copolymer

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110

vesicles, diblock copolymer vesicles of 200 nanometres in diameter were

produced to encapsulate a pH sensitive dye, pyranine. This system was

used in order to prove the membrane incorporation of gramicidin. The

successful accomplishment of the ion-channel incorporation is an

important step towards the design of pH-triggerable nanoreactors and

artificial organelles, where the activity of the protected active compounds

can be activated or deactivated via specific ion-channels “on demand”.

iii) Next to the controlled activity, the site specific action of the active

compound is also important. To address this challenge, CuII-trisNTA-

functionalised PB-b-PEO polymersomes were used to study molecular

recognition process of specific peptide binding. A model system of an

oligohistidine sequence consisting of six histidines – a His6-tag – was

used to study their binding to copper centres on the polymersome

surface. Upon binding, a detailed structural view of the rearrangement of

the metal coordination sphere was obtained. More than 200 fluorescently

labelled His6-tags were bound to one polymersome, proving efficient

binding. The system was also characterised by a high binding affinity.

Different EPR and ENDOR methods were used to investigate the

electronic and geometric change of the copper centre complexed in the

NTA pocket upon binding of the model His6-tags. These experiments, as

well as DFT computations, confirmed strong and specific binding. This

detailed knowledge about the molecular recognition behaviour of the

system and the simple preparation procedure allows to further engineer

specific artificial organelles with site-specific action capabilities.


111

6. Experimental section

Materials

Cu/Zn-superoxide dismutase (Cu/Zn-SOD) from bovine erythrocytes

(>3,000 U/mg), lactoperoxidase (LPO) from bovine milk (>200 U/mg),

catalase (CAT) from bovine liver (≥10,000 units/mg), xanthine oxidase

(XO) (microbial, 7 U/mg), xanthine (XA), sodium phosphate buffer (DPBS

1 x Dulbecco`s salts without calcium chloride and magnesium chloride,

pH 7.3), hydrogen peroxide, 30% (w/w)) and dimethyl sulphoxide (DMSO)

(>99.5%),were purchased from Sigma Aldrich and were used without any

further treatment. 10-acetyl-3,7-dihydroxyphenoxazine (amplex red),

AlexaFluor 488 carboxylic acid succinimidyl ester, and AlexaFluor-633

carboxylic acid succinimidyl ester were obtained from Molecular Probes

(Invitrogen) (Eugene, OR, USA) and were used as received. Dylight-488

carboxylic acid succinimidyl ester and dylight-633 carboxylic acid

succinimidyl ester were purchased from Thermo Fisher Scientific Inc.

(Rockford, IL, USA) and were used without further purification. Amplex

red stock solutions were prepared in DMSO (50 mM) and stored at -20

C°. N-Octyl-oligo-oxyethylene (OPOE) was purchased from Enzo Life

Sciences (Lausen, Switzerland). Hoechst and propidium iodide were

purchased from Invitrogen. An ROS detection kit from Enzolife,

polycarbonate membrane filters (Millipore), and sepharose 2B (Sigma

Aldrich) were used. RPMI-1640 cell culture medium (GIBCO), fetal calf

serum (FCS), penicillin and streptomycin (Sigma Aldrich) and PBS

(GIBCO) were used in the cell experiments. L-α-phosphatidylcholine,

gramicidin from bacillus brevis (a mixture of gramicidin A, B, C and D),

pyranine (8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt), and acid

phosphatase were purchased from Sigma-Aldrich and used without

further purification. ELF97 phosphatase substrate was purchased from

Invitrogen. 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium

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112

hexafluorophosphate (HCTU) and rink amide AM resin (0.61mmol/g)

were purchased from IRIS Biotech GmbH, Marktredwitz, Germany. The

amino acids (N-α-t.-Boc-L-histidine) were obtained from Novabiochem,

Läufelfingen, Switzerland. Sulphorhodamie B acid chloride (λex: 560 nm,

λem: 580 nm) and copper(II)triflate was purchased from Sigma-Aldrich,

Buchs, Switzerland. Dichlormethane was provided by Brenntag

Schweizerhall AG, Basel, Switzerland, dimethylformamide (DMF) by J. T.

Baker and acetonitrile (ACN) by Fischer Scientific. DMF was treated with

aluminium oxide to reduce the concentration of free amines prior to

application in peptide synthesis.

Methods used for the design of antioxidant nanoreactors/artificial

peroxisomes

Outer membrane protein F production

OmpF expression was carried out in E. coli (BL21 (DE3)omp8)72

according to a previously optimised protocol53 and the product was stored

at 700 µg/mL with 3 wt% OPOE stock solution at 4 C°.

Fluorescence labelling of SOD, LPO and CAT

SOD was labelled with AlexaFluor-488 carboxylic acid succinimidyl

ester, LPO was labelled with AlexaFluor-633 or dylight-633 carboxylic

acid succinimidyl ester and CAT was labelled with dylight-488 carboxylic

acid succinimidyl ester following the protocol provided by Invitrogen

(www.invitrogen.com). The solutions containing labelled enzyme were

filtered through a 0.22 μm Millex low protein-binding Durapore membrane

(Millipore, Billerica, MA, USA) and separated by fast protein liquid

chromatography (FPLC) on a Sephadex G25 (Aldrich) column

equilibrated with phosphate-buffered saline buffer (PBS). The

concentration of labelled enzyme and the degree of labelling was

determined by dual wavelength FPLC (Äkta FPLC, GE Healthcare

http://www.invitrogen.com/


113

Europe GmbH, Glattbrugg, Switzerland) or by UV/VIS. After the labelling

and purification procedure, 209.56 µM of labelled SOD, 4.45 µM of

labelled LPO and 20 µM of labelled CAT were obtained.

PMOXA-PDMS-PMOXA vesicles preparation

Synthesis and characterisation of ABA triblock copolymer PMOXAn-

PDMSm-PMOXAn (n = 12 and m = 55) has been described previously.53

Vesicles were prepared at room temperature by drop-wise addition of

PBS to ABA copolymer under continuous stirring, obtaining a 5 mg/mL

polymer concentration. The aqueous polymer solution was extruded with

an Avanti mini-extruder (Avanti Polar Lipids, Alabama, USA) through a

400 nm diameter pore-size polycarbonate (PC) membrane (one time),

and through a 200 nm pore-size PC membrane (11 times) in order to

reduce size polydispersity of the vesicles.

Preparation of nanovesicles containing individual enzyme types and

SOD/LPO (or SOD/CAT) containing artificial peroxisomes

To encapsulate an individual enzyme type in vesicles, a solution of

SOD, LPO or CAT (in concentrations between 0.1 – 0.83 mg/mL) were

mixed with a solution of ABA block copolymer, followed by the

preparation steps described above. A mixture of SOD and LPO at

different ratios in PBS was added drop-wise to the ABA polymer solution

to encapsulate both enzymes simultaneously. When required, channel

protein68 was simultaneously incorporated by drop-wise addition of the

OmpF porin stock solution to the enzyme(s)-ABA copolymer mixture with

continuous stirring to reach a final concentration of 50 µg/mL OmpF. Non-

encapsulated enzymes were separated from vesicles by 48 hour dialysis

(Spectrapore dialysis tube, mean width cut-off size: 300 kDa, Spectrum

Laboratories Inc.), exchanging dialysis PBS buffer after 24 hours.

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Transmission electron microscopy (TEM)

Empty vesicles, vesicles containing a single-enzyme constituent, SOD-

AlexaFluor-488, LPO-AlexaFluor-633 or CAT-dylight-488, vesicles

containing co-encapsulated SOD-AlexaFluor-488 and LPO-AlexaFluor-

633 and cell-samples were negatively stained with 2% uranyl acetate

solution and deposited on carbon-coated copper grids. Vesicle samples

were studied using a transmission electron microscope (Philips Morgagni

268D) at 293 K.

Light scattering (LS)

Dynamic (DLS) and static (SLS) light-scattering experiments were

performed on an ALV (Langen, Germany) goniometer, equipped with an

ALV He-Ne laser (JDS Uniphase, wavelength λ = 632.8 nm). Vesicles

dispersions were prepared by serial dilution to polymer concentrations

ranging from 2.5 to 0.05 g/L. Light scattering was measured in 10 mm

cylindrical quartz cells at angles of 40 − 150° at 293 ± 0.5 K. The

refractive index increment ∂n/∂C of the vesicle dispersions was

determined to 0.16 0.02 mL/g at 632.8 nm with an ALV-DR1 differential

refractometer. The photon intensity auto-correlation function g2(t) was

determined with an ALV-5000E correlator (scattering angles between 40°

and 150°). DLS data were analysed via non-linear decay-time analysis

supported by regularized inverse Laplace transform of g2(t) (CONTIN

algorithm). The angle-dependent apparent diffusion coefficient was

extrapolated to zero momentum transfer (q²) using ALV Correlator

software static and dynamic FIT and PLOT 4.31. Angle and

concentration-dependent SLS data were analysed using a Guinier plots

approach.


115

Fluorescence correlation spectroscopy (FCS) and fluorescence cross-

correlation spectroscopy (FCCS)

FCS and FCCS measurements were performed at room temperature

on a Zeiss LSM 510-META/Confcor2 laser-scanning microscope

equipped with an argon2-laser (488 nm), a helium/neon laser (633 nm)

and a 40× water-immersion objective (Zeiss C/Apochromat 40X, NA 1.2)

or a 63x water emulsion lens (Olympus). The pinhole was adjusted to 70

µm, and 90 µm, respectively. For each measurement, 10 μL of sample

solution, containing either free dye AlexaFluor-488 or AlexaFluor-633 or

dylight-488 or dylight-633, each concentrated to 50 nM, or containing an

enzyme- (or combined enzyme-) encapsulated vesicle solution used

directly after the purification step, were placed in special, chambered

quartz glass holders (Lab-Tek; 8-well, NUNC A/S). For FCS, spectra were

recorded over 30 s, and each measurement was repeated 10 times.

Excitation power of the argon2 laser was PL = 15 mW, and the excitation

transmission at 488 nm was 5%. Excitation power of the helium/neon

laser was PL = 5 mW, and excitation transmission at 633 nm was 1%.

Diffusion times for free dye (AlexaFluor-488, AlexaFluor-633, dylight-488

and dylight-633) as well as for labelled SOD, labelled LPO and labelled

CAT were independently determined and fixed in the fitting procedure.

The results were presented as a mean value of three independent

measurements. For the fitting of the autocorrelation function according to

a two-component model, the following equation 8 was used:

(8)

where,

N: number of fluorescent particles

S: structural parameter

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τD1: diffusion time of the component 1 in the assay.

1-Y: fraction of particles with diffusion time τD1

τD2: diffusion time of component 2 in the assay.

Y: fraction of particles with diffusion time τD2

f(T): function used for fitting the tripled characteristics τT and %

τT of the fluorescent label within the assay.

By means of an iterative least-square method, the values calculated

by the algorithm are compared repeatedly to the experimentally

generated autocorrelation curve and approximated until the difference

between the two curves is minimised.

FCCS measurements were performed on the same instrument in the

FCCS mode. Overlap of the confocal volumes was ensured in each

channel by optimising pinhole settings in each measurement channel and

performing calibration measurements. Cross-talk was measured

independently in each detection channel to exclude false-positive cross-

correlation.171 The fraction of co-encapsulated enzymes (R) can be

estimated by the formula (equation 9):

(9)

where Rg is the relative autocorrelation amplitude of the green channel,

Rr: relative autocorrelation amplitude of the red channel, and

Rcc: the relative amplitude of the cross-correlation.

The theoretical fraction of co-encapsulated enzymes, P, in the

hypothesis of independent event probabilities p1 (fraction of encapsulated


117

SOD) and p2 (fraction of encapsulated LPO) can be estimated by the

formula (equation 10):

(10)

Individual enzyme types activity assay

LPO and CAT kinetics in solution or in nanovesicles were studied with

a PerkinElmer LS55 fluorescence spectrometer (Waltham,

Massachusetts, USA) at ambient temperature. Free LPO kinetics was

tracked using a gradient of H2O2 and amplex red as follows:

concentrations from 0.3 μM to 2.4 µM H2O2, 5 nM LPO solution in PBS

buffer and concentrations from 0.47 M to 3.75 μM amplex red. The

kinetics of LPO in nanovesicles were followed immediately after mixing

H2O2 (1.09 – 5.86 μM) with a 50-fold diluted LPO-containing nanovesicle

solution and the addition of amplex red (0.18 – 1 M).

The solutions were mixed in a quartz cuvette and the fluorescence

intensity of resorufin (ex = 530 nm, em = 580 nm), as the reaction

product, was recorded.

CAT activity was compared to LPO activity by a competition reaction

as presented in Scheme 1.

Scheme 1 CAT activity assay. Reprinted with permission from Ref. 78. Copyright

2013 American Chemical Society.

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Multi-enzyme activity assay

The cascade reaction of SOD and LPO in tandem was followed in free

solution or in nanovesicles by fluorescence spectroscopy using the set-up

of FCS. Activity tests of free SOD and LPO in sequence were performed

by mixing SOD (0.2 nM - 66 µM) with LPO (0.016 - 1µM) in different

concentration domains and in the presence of amplex red (9 µM) with

various amounts of xanthine (2 µM – 25 μM). The cascade reaction was

started by the addition of xanthine oxidase (0.002 − 0.17 U/mL), with the

concentrations adjusted to not short circuit SOD due to generation of

H2O2 by the xanthine-xanthine oxidase system. Resorufin formation was

followed as count rate (kHz).

Activity tests of SOD/LPO co-encapsulated vesicles were carried out

by mixing 10 µL SOD-LPO-containing nanovesicle solution with amplex

red (23.8 µM), and xanthine (35.71 µM) to a final volume of 210 μL. The

cascade reaction was started by the addition of xanthine oxidase

(0.24 U/mL).

Cell experiments

HeLa cells were cultured and maintained in Modified Eagle's Medium

(DMEM) supplemented with high glucose, 10% fetal calf serum, 1%

penicillin (10`000 units/mL) and streptomycin (10`000 mg/mL) (purchased

from Sigma Aldrich), 2 mM L-glutamine and 1 mM pyruvate. Cells were

grown in a humidified incubator (HERA Cell 150, Thermo Scientific,

Germany) at 37 °C in a 5% CO2 atmosphere until a confluence of 60 –

80% was achieved, and then harvested by trypsin-EDTA.

Cytotoxicity of the vesicles and APs

Cells were cultured in a 96-well plate at a density of 2 x 104 cells/well,

and incubated with 100 µL of DMEM growth medium per well for 24 h.

Different concentrations of empty polymer vesicles and enzyme-loaded


119

polymer vesicles were added to different wells and incubated with cells

for a period of time (24 and 48 h). Subsequently, 20 µL of CellTiter® 96

Aqueous One Solution Cell Proliferation Assay (MTS) reagent (Promega)

were added to each well and then incubated for a further 2 h. The

absorbance of each well mixture was measured with a Spectromax M5e

microplate reader (Molecular Devices) at 490 nm.

Cell uptake of APs

HeLa cells were cultured in a 6-well plate at different densities of cells

per well (1 x 105 for CLSM, 2 x 105 for flow cytometry analysis, and 1 x

106 for TEM) in DMEM medium and incubated with enzyme-loaded

polymer vesicle solution at different time intervals (2-, 4-, 8-, 18-, 24 h) at

37 °C in a humidified CO2 incubator. For flow cytometry analysis, cells

incubated with a solution of enzyme-loaded polymer vesicles were

washed with PBS to remove free enzyme-containing vesicles and

quantitatively analysed for rate of uptake of enzyme-containing vesicles

with a flow cytometry set-up (CYAN, BD biosciences). A total of 10,000

events were analysed for each condition.

Study of APs escape from endosome

HeLa cells pre-incubated with a polymer vesicle solution containing co-

encapsulated SOD and LPO were fixed with a solution of 3%

paraformaldehyde in PBS, and 0.5% glutaraldehyde in PBS containing

1.5% saccharose (pH 7.3), and then post-fixed in 0.5% aqueous osmium

tetroxide. The samples were then dehydrated using a graded series of

ethanol solutions, block-stained in 6% of uranyl acetate and embedded in

LR white resin (Sigma Aldrich). Ultra-thin sections were contrasted with

0.3% lead citrate and imaged with TEM.

For CLSM visualisation, the HeLa cell culture was first treated with

enzyme-loaded polymer vesicles (100 μg/mL) for 4 h, followed by

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treatment with pH-rodo™ Red dextran (20 μg/mL) for a further 20 min at

37 °C following the supplier's protocol. The cells were rapidly washed with

PBS and prepared for imaging.

Cellular ROS detoxification by APs

HeLa cells were treated with paraquat to induce oxidative stress and

analysed by flow cytometry (CYAN, BD Biosciences). HeLa cells at a

density of 1 x 105 plated in a 6-well plate were first incubated with

enzyme-loaded polymer vesicles (500 µg/mL) for 24 h, and subsequently

with paraquat (2 mM) for a further 24 h at 37 °C in a 5% CO2 atmosphere.

Cells were washed with PBS buffer and stained with propidium iodide (PI,

50 mg/mL) to determine cell viability. A total of 10,000 events were

analysed for each condition. To analyse the ROS detoxification kinetics of

artificial peroxisomes in live cells, HeLa cells were seeded at 4 x 103

cells/well in a 96-well plate incubated overnight for cell attachment,

followed by incubation with APs vesicles for 8 h. Afterwards, cells were

treated with ROS/superoxide detection mix from Enzolife’s

ROS/superoxide detection kit for 1 h. Cells were then incubated with

pyocyanin (300 µM) to induce ROS generation in the cells. ROS

detoxification kinetics was measured with a Spectromax M5e microplate

reader (Molecular Devices) in the kinetic mode with a 550/620 nm filter

set.

Methods used for the design of pH-triggered nanoreactors

PMOXA-PDMS and L-α-phosphatidylcholine vesicles preparation and

encapsulation of the pH-sensitive dye (pyranine)

PMOXA14-PDMS65 was used to prepare vesicles at room temperature

by using the film hydration method obtaining a 5 mg/mL polymer

concentration. The direct dissolution method was used for the preparation


121

of vesicles based on L-α-phosphatidylcholine. In order to encapsulate the

pH-sensitive dye pyranine, the polymer film or the lipids were hydrated

using pyranine dissolved in PBS at a concentration of 50 mM. The

aqueous polymer solution and liposome solution were extruded with an

Avanti mini-extruder (Avanti Polar Lipids, Alabama, USA) through a

400 nm diameter pore-size polycarbonate (PC) membrane (one time),

and through a 200 nm pore-size PC membrane (11 times) in order to

reduce size polydispersity of the vesicles.

Measurements of gramicidin incorporation by monitoring pH change inside

vesicles and design of pH-sensitive nanoreactors

Functional gramicidin ion-channel formation in the liposome or

polymersome membrane was measured by using a PerkinElmer

fluorescence spectrometer LS55 (Waltham, Massachusetts, USA) in the

time driven mode. Pyranine encapsulated polymersomes were diluted

1:300. A pH change was monitored during addition of HCL to lower the

pH from pH 8.3 to 5. The effect of subsequent addition of a low amounts

of gramicidin (dissolved in different suitable solvents such as dimethyl

sulphoxide (DMSO), trifluoroethanol (TFE), dioxane, pyridine,

isoporpanol, decane and the mixtures DMSO:ethanol (DMSO:EtOH) in

the ratio 1:1 and the mixture TFE:DMSO:EtOH (2:1:1)) to yield

concentrations between 30 nM and 130 nM was compared in means of

the change of the fluorescence intensity of pyranine inside the vesicles to

the condition when the same amount of solvent was added and when no

further components were added. In order to characterise pH-sensitive

nanoreactors, simultaneously acid phosphatase (10 μM) and ELF97 (12.5

μM) were added during the vesicle formation process and purified as

described above. pH-dependent activity of acid phosphatase inside the

vesicles was measured using also the LS55 fluorescence spectrometer in

the time driven mode.

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Pyranine-encapsulated vesicles in condition of pHinside = pHoutside,

pHinside < pHoutside and after addition of gramicidin were negatively stained

with 2% uranyl acetate solution and deposited on carbon-coated copper

grids. Vesicle samples were studied using a transmission electron

microscope (Philips Morgagni 268D) at 293 K.

Light scattering (LS)

Dynamic (DLS) experiments were performed on an ALV (Langen,

Germany) goniometer, equipped with an ALV He-Ne laser (JDS

Uniphase, wavelength λ = 632.8 nm). Light scattering was measured in

10 mm cylindrical quartz cells at a fixed angle of 90° at 293 ± 0.5 K for

comparison of the effects of pH change and gramicidin incorporation in

the polymersome membrane. The photon intensity auto-correlation

function g2(t) was determined with an ALV-5000E correlator. DLS data

were analysed via non-linear decay-time analysis supported by

regularized inverse Laplace transform of g2(t) (CONTIN algorithm).

Methods used for study molecular interaction of metal-functionalised

polymersomes with His-tags

Polymers

Synthesis and characterisation of the diblock copolymer PB39-b-PEO36

was described previously.21 A mixture of PB39-b-PEO36-SA-OH containing

10% PB39-b-PEO36-SA-trisNTA.d-Cu2+ was used for all experiments. The

diblock copolymer is functionalised with trisNTA a variant of NTA and

complexed with Cu2+ ions. The non-functionalised diblock copolymer was

characterised at a molecular mass of 3530 g/mol, which increased to

3690 g/mol upon functionalisation with trisNTA. Both block copolymers

have a low polydispersity index.21


123

Peptide synthesis and fluorescent labelling

The peptides were synthesised on a Syro I Peptide Synthesiser

(MulitSynTech GmbH, Witten, Germany) using solid phase synthesis with

an Fmoc-strategy. HCTU as the coupling reagent, with N-ethyldi-

isopropylamine (DIPEA) dissolved in N-methyl-2-pyrrolidone (NMP) and

used as the base to couple a-N-Fmoc-protected amino acids to the rink

amide resin. For elongation, Fmoc-His-OH (0.5 mol/L, 4 equiv.), HCTU

(0.5 mol/L, 4 equiv.) dissolved in DMF, and DIPEA (12 equiv.) were

added to the resin. The mixture was agitated for 1 h and washed with

DMF (3 x 3 mL). Fmoc deprotection was performed with 20% piperidine in

DMF followed by 3 min agitation, draining, and repetition of deprotection

for 10 min. Resin was subsequently washed with DMF (5x3 mL).

Acetylation of unreacted amine groups was performed following each

coupling with a solution of acetic anhydride/DIPEA (3 mol/L, 5 equiv.) in

DMF. After synthesis, the peptide resin was alternately washed with DMF

(3 x 6 mL), isopropanol (3 x 6 mL), and dichloromethane (3 x 6 mL), and

then dried under vacuum.

The coupling of the dye to the N-terminus was performed on peptide

still bound to the solid phase. A DMF solution of carboxylic-acid-

succinimidyl-ester-activated dye (sulphorhodamine B) was added to the

peptide. Uncoupled dye was washed away alternating with DMF (3 x

6 mL), isopropanol (3 x 6 mL), and dichloromethane (3 x 6 mL).

Cleavage of the peptide from the resin and removal of its protective

groups was performed with a mixture of 95% trifluoroacetic acid (TFA),

2.5% tri-isopropylsilane, and 2.5% H2O. The resin was additionally

washed two times with this mixture (2 mL) and the peptide was

precipitated in 40 mL cold di-isopropylether (IPE). The precipitated

peptide was washed three times with cold IPE by centrifugation and

decantation, before being dried under vacuum.

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Peptide purification and characterisation

Purification and analysis of the peptides were performed by HPLC

(Shimadzu Prominence 20A, Japan) on reverse phase (RP) columns

(Merck Chromolith, RP-18e, 100 mm x 10 mm and 100 mm x 4.6 mm).

The separation of the unlabelled peptide from labelled peptide yielded

sample purities higher than 95%. This was detected by absorption at

280 nm and 560 nm. Linear gradients of solvent A (ACN) and solvent B

(0.1% TFA, or 2% acetic acid in bidistilled water) were used.

The eluted sample was collected in fixed volume fractions, which were

subsequently analysed for purity and molecular weight using HPLC and

matrix-assisted laser desorption/ionisation time-of-flight mass

spectroscopy (MALDI-TOF-MS). MALDI-TOF-MS was performed for

mass confirmation with a Voyager-DETM System (Applied Biosystems,

USA) in positive reflector mode, an accelerating voltage of 25 kV, grid

voltage of 75% and 100 − 300 ns extraction delay time. Matrix (α-cyano-

4-hydroxycinnamic acid) sample mixtures were prepared using a solution

of ACN/0.1% TFA in bidistilled water on a 100-well gold plate. Fractions

with matching mass and exceeding 95% purity were pooled with

ammonia before neutralisation and then lyophilised. Products were stored

under argon at -18 °C.

PB-b-PEO vesicle preparation and His6-tag binding.

PB-b-PEO vesicles were prepared (800 µM) according to the film

rehydration method and contained 10% CuII-trisNTA functionalised PB-b-

PEO copolymer.21 His6-tags or sulphorhodamine B acid chloride labelled

His6-tags (s-His6-tags) were added to the vesicle solution at different

concentrations depending on the used measurement technique (see

below). The solutions were extruded with an Avanti mini-extruder (Avanti

Polar Lipids, Alabama, USA) through a 400 nm diameter pore-size


125

polycarbonate (PC) membrane (one time), and through a 200 nm pore-

size PC membrane (11 times) in order to minimize nanovesicle size

distribution.

Dynamic and Static Light Scattering (DLS and SLS)

Light scattering experiments were performed on an ALV (Langen,

Germany) goniometer, equipped with an ALV He-Ne laser (JDS

Uniphase, wavelength = 632.8 nm). Vesicle dispersions were prepared

by serial dilution to polymer concentrations ranging from 0.7 to 0.1 g/L.

Light scattering was measured in 10 mm cylindrical quartz cells at angles

of 30 – 150° at 293 K ± 0.5 K. The photon intensity auto-correlation

function g(t) was determined with an ALV-5000E correlator (scattering

angles between 30° and 150°). DLS data were analysed via non-linear

decay-time analysis supported by regularized inverse Laplace transform

of g(t) (CONTIN algorithm). The angle-dependent apparent diffusion

coefficient was extrapolated to zero momentum transfer (q²) using ALV

Correlator software static and dynamic FIT and PLOT 4.31.


TEM images were taken using a transmission electron microscope

(Philips Morgagni 268D) operated at 80 keV. The vesicle samples diluted

to a final polymer concentration of 8 µM were negatively stained with 2%

uranyl acetate solution for 10 s and deposited on carbon-coated copper

grids in order to perform the measurement.

Confocal laser-scanning micorscopy (CLSM)

PB-b-PEO vesicles were prepared (800 µM) as mentioned above and

were incubated with fluorescently labelled His6-tags (s-His6-tags) (1 µM)

for 5 min at room temperature. The solution was dialyzed for 24 h with a

Spectrapore dialysis tube (mean width cut-off size: 300 kDa, Spectrum

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Laboratories Inc.) to separate non-bound s-His6-tags from vesicles. This

solution was investigated at room temperature in special chambered

quartz glass holders with a confocal laser-scanning microscope (Zeiss

LSM 510-META/Confcor2), in LSM mode, using a Helium/Neon laser

(543 nm) and a 63× water-immersion objective (Zeiss C/Apochromat

63X, NA 1.2). HeNe laser excitation power was PL = 1 mW, and

excitation transmission at 543 nm was 20%.

Fluorescence Correlation Spectroscopy (FCS)

For FCS measurements, PB-b-PEO vesicle solutions with different CuII-

functionalised PB-b-PEO concentrations (ranging from 2 µM to 8 µM)

were prepared as mentioned above. The vesicle solutions containing

different amounts of copper were incubated with 50 nM s-His6-tags and

were immediately measured without dialyzing. FCS measurements were

performed at room temperature in special chambered quartz glass

holders (Lab-Tek; 8-well, NUNC A/S), on the Zeiss LSM 510-

META/Confcor2 laser-scanning microscope equipped with a HeNe laser

(543 nm) and a 40× water-immersion objective (Zeiss C/Apochromat 40X,

NA 1.2), with pinhole adjusted to 78 µm. Spectra were recorded over

30 s, and each measurement was repeated 10-times. The excitation

power of the HeNe laser was PL =1 mW, and the excitation transmission

at 543 nm was 10%. The structural parameter and diffusion times of the

free dye (sulforhodamine B acid chloride) and of the dye-labelled His6-

tags were independently determined and fixed in the fitting procedure.

These parameters were used to fit the autocorrelation curve of s-His6-tags

bound PB-b-PEO vesicles. The fluorescence signal was measured in real

time and the autocorrelation function was calculated by a software

correlator (LSM 510 META - ConfoCor 2 System). The results were

presented as a mean value of three independent measurements.


127

X-band CW-EPR

Samples for X-band CW-EPR measurements were prepared similar to

FCS with the exception that an 800 µM vesicle solution was incubated

with 2.0 mM s-His6-tags. X-band CW-EPR measurements (microwave

(MW) frequency of about 9.44 GHz) were performed on a Bruker ESP

300E instrument, equipped with a liquid Helium cryostat (Oxford Inc.). A

MW power of 10 mW, a modulation frequency of 100 kHz and a

modulation amplitude of 0.5 mT were applied. All spectra were taken at

100 K and 10 K. The EasySpin program was utilized to simulate the CW-

EPR spectra.172

X-band pulsed EPR experiments were performed on a Bruker Elexsys

spectrometer (9.76 GHz), equipped with a helium-gas flow cryostat

(Oxford Inc.). The measurements were done at 4 K, with a repetition rate

of 1 kHz.

ENDOR experiments

The ENDOR experiments were performed using the MW pulse

sequence π – T – π/2 – – π – – echo, whereby a π radio-frequency (rf)

pulse is applied during time T. The following parameters were used: tπ =

48 (96) ns, tπ/2 = 24 (48) ns, T = 15 µs, trf = 13 µs and = 200 ns. The

Mims ENDOR experiments were performed with the MW pulse sequence

– π/2 – τ – π/2 – T – π/2 – τ – echo. The parameters used were: tπ/2 =

16 ns, T = 15 µs, trf = 13 µs. The spectra were recorded at = 104 ns.

HYSCORE

The standard HYSCORE experiments were performed using the π/2 – τ

– π/2 – t1 – π –t2 – π/2 – τ – echo sequence of pulses with tπ/2 = 16 ns

and tπ = 16 ns. The time intervals t1 and t2 were varied in steps of 16 ns.

In order to eliminate unwanted echoes, a four-step phase cycle was

applied. The time-domain HYSCORE spectra were base-line corrected

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with a third-order polynomial, apodized with a Hamming window and zero

filled. The absolute-value spectra, obtained after two-dimensional Fourier

transformation, were added to different τ values (104 ns, 120 ns, 176 ns)

to reduce the blind-spot effects. The HYSCORE spectra were simulated

using a program developed at ETH Zurich.173

DFT computations

Spin-restricted DFT computations were performed with the ORCA

package174-177 on different models for aqueous CuII-NTA and CuII-NTA-

His6-tag binding (see results and discussion sections). Geometry

optimisations were performed with the BP86 functional with an Ahlrich

split-valence plus polarization (SVP) basis set for all other atoms except

copper, for which a more polarized triple zeta valence basis set (TZVPP)

is used. The geometry optimisations were tested for different starting

geometries and the most stable geometry overall was chosen for further

computation of the EPR parameters. For the latter, the B3LYP functional

was taken, combined with the EPR-II basis set for nitrogen and hydrogen,

the ORCA basis set “Core Properties” (CP(PPP)) and SVP for all other

atoms. A dielectric surrounding with the dielectric constant of water was

simulated assuming the COSMO model in all computations.


129

7. Appendix

Calculation of the encapsulation efficiency:

To determine maximum enzyme encapsulation efficiency based on the

number of encapsulated enzymes per vesicle, following equations were

used:

(11)

(12)

(13)

( ) (14)

Where, in equation 11, c is the enzyme concentration [gmol-1] used for

encapsulation and NA the Avogadro number. The reciprocal value of

equation 11 gives the apparent volume per particle Vapparent (equation 12).

The number of particles N*enzymes is derived by dividing the volume of the

polymer vesicles Vvesicle by the volume of apparent volume Vapparent,

representing the theoretical encapsulation of enzymes by one polymer

vesicle (equation 13). To calculate the encapsulation efficiency, EEv (%),

the number of particles determined by FCS brightness measurements,

N*exp, is divided by the theoretical value (equation 14).

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Coordinates of the used models for DFT and spin density distributions:

Model A: [Cu(N(CH2COO)2(CH2COOH))(H2O)2]

Fig. 54 Calculated spin density of model A. The contour levels are set at 0.0017 and

-0.0017, respectively. Blue represents positive spin density, while red represents

negative spin density.

Model A: Coordinates (in Å)

H 1.986319 -3.766505 -0.002538

H 1.048407 -5.276164 -0.082316

C 0.954631 -4.173606 0.006035

C 0.363875 -3.792742 1.372461

O -0.360862 -2.715521 1.387576

H 1.911532 -3.810738 -2.437720

H 0.545170 -2.868100 -3.101400

H -1.136889 -4.164111 -2.675405

H -0.208779 -5.532695 -1.986454

N 0.206808 -3.604295 -1.148356

Cu -0.780084 -1.904933 -0.339569

O -1.742134 -1.379114 -2.071862

O -1.564094 -0.255161 0.566701

C -0.729385 -4.584561 -1.732349

C 1.130284 -3.063877 -2.179103

C 1.798449 -1.725157 -1.756243

O 2.872735 -1.424390 -2.313065

O 1.144457 -1.027523 -0.904697


131

C -1.943043 -4.877461 -0.858495

H -3.145692 -6.276150 -0.421447

O -2.310240 -6.163282 -0.933767

O -2.542502 -4.026354 -0.216120

O 0.638868 -4.455462 2.380576

H -2.597000 -0.945433 -1.865298

H -1.557722 -0.397582 1.537494

H -1.227656 -0.678462 -2.526009

H -0.959451 0.506383 0.438370

Model B: [Cu(N(CH2COO)2(CH2COOH))(C11H15N5O)]

Fig. 55 Calculated spin density of model B. The contour levels are set at 0.0017 and



Model B: Coordinates (in Å)

O 0.602432 -4.366001 2.366477

C 0.350746 -3.697671 1.351770

O 1.322347 -1.035162 -1.016393

C 1.913262 -1.808572 -1.848316

H -4.370836 2.857339 -1.736942

H -4.326905 2.165665 -3.798643

O -0.303207 -2.579311 1.353159

H -1.394634 1.904801 -4.778039

H -2.787056 1.154668 -5.602032

C -2.401450 1.473803 -4.610637

H -0.173716 0.207722 -2.900534

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132

H -0.104274 1.294144 -0.635806

C -0.986255 1.165798 -0.000845

C -2.900475 -1.442676 -2.439612

Cu -0.678248 -1.780782 -0.419280

N -1.608280 -1.242648 -2.165215

N -1.393501 -0.085058 0.424616

C -1.199137 -0.172971 -2.944475

C -2.264897 0.291787 -3.699694

N -3.324982 -0.540675 -3.361746

H -4.270859 -0.505503 -3.752520

H -3.546140 -2.202240 -1.985470

C -2.478165 0.098388 1.179757

N -2.777760 1.424239 1.244256

C -1.846837 2.132198 0.494726

H -3.062747 -0.681864 1.679489

H -3.558600 1.821479 1.774000

C -3.328439 2.578944 -4.049666

C -2.718276 3.329173 -2.860547

N -3.476061 3.350105 -1.727732

C -3.131937 4.107925 -0.528749

C -1.882071 3.605736 0.240401

H -4.022616 4.086288 0.128643

H -2.954803 5.170926 -0.800015

H -3.490140 3.335766 -4.846359

O -1.618273 3.897314 -2.942389

H -1.825320 4.179835 1.190601

H -0.979793 3.857812 -0.348261

C 1.154341 -3.114166 -2.228934

N 0.202492 -3.572055 -1.184932

O 3.001551 -1.603611 -2.426269

H 0.575332 -2.895652 -3.149794

C -0.782080 -4.516595 -1.745364

H -1.160684 -4.103560 -2.703868

H -0.316056 -5.500125 -1.970811

C 0.912737 -4.150743 -0.010429

H 1.886217 -3.913216 -2.477798

C -2.013151 -4.720475 -0.869047

O -2.579433 -3.831286 -0.248680

O -2.442060 -5.990694 -0.915201

H -3.283128 -6.049242 -0.403119

H 0.940642 -5.259686 -0.063526

H 1.967184 -3.806616 -0.030207


133

Model C: [Cu(N(CH2COO)3)(C11H15N5O)]-

Fig. 56. Calculated spin density of model C. The contour levels are set at 0.0017 and



Model C: Coordinates (in Å)

O 0.926091 -4.315926 2.321426

C 0.558879 -3.679574 1.316592

O 1.263008 -1.006906 -1.041422

C 1.838176 -1.749821 -1.904118

H -4.327253 2.837797 -1.746011

H -4.291118 2.170098 -3.818731

O -0.125606 -2.583357 1.359648

H -1.350400 1.875660 -4.758340

H -2.737551 1.131332 -5.595999

C -2.363172 1.454281 -4.601453

H -0.202956 0.239622 -2.765101

H -0.072479 1.178235 -0.422051

C -1.014227 1.088705 0.129711

C -2.912779 -1.466237 -2.445785

Cu -0.802761 -1.886316 -0.363633

N -1.643377 -1.235327 -2.106224

N -1.492393 -0.146972 0.523713

C -1.216786 -0.160207 -2.865571

C -2.250859 0.278299 -3.679181

N -3.310202 -0.575318 -3.392913

H -4.236075 -0.557271 -3.829340

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134

H -3.551224 -2.241888 -2.009024

C -2.641561 0.075919 1.161082

N -2.914065 1.411203 1.184595

C -1.894395 2.085042 0.524156

H -3.298664 -0.682647 1.601380

H -3.733103 1.834538 1.629047

C -3.287239 2.573842 -4.063300

C -2.682454 3.326981 -2.873982

N -3.439690 3.343427 -1.740875

C -3.090379 4.095822 -0.540404

C -1.872386 3.556720 0.253649

H -3.992548 4.110910 0.101781

H -2.873153 5.149525 -0.817587

H -3.431829 3.325748 -4.867958

O -1.586170 3.902764 -2.954894

H -1.812098 4.140558 1.198383

H -0.950427 3.771309 -0.319655

C 1.117492 -3.082877 -2.274411

N 0.238591 -3.607442 -1.212457

O 2.897574 -1.501980 -2.529977

H 0.490583 -2.869658 -3.165252

C -0.796413 -4.541126 -1.689452

H -1.120777 -4.206244 -2.697008

H -0.393004 -5.573851 -1.796641

C 1.010544 -4.159346 -0.076019

H 1.883745 -3.830852 -2.582332

C -2.091358 -4.602167 -0.821057

O -2.434265 -3.524442 -0.230012

O -2.724058 -5.685285 -0.838941

H 1.015169 -5.271075 -0.095836

H 2.070530 -3.841981 -0.169094


135

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151

9. Acknowledgements

I cordially thank Prof. Dr. Cornelia Palivan for giving me all the

important support and the necessary possibilities to intensively work this

thesis out. I also thank Prof. Wolfgang Meier giving me the possibility to

work on the projects in his group.

I thank Dr. Vimalkumar Balasubramanian for all his efforts during our

collaboration in the group and beyond that. Especially, I appreciate this

power in performing all the important cell experiments and giving me

advice. I also owe Prof. Sabine Van Doorslaer and Dr. Maria Ezhevskaya

a debt of gratitude for successful collaboration on EPR experiments. In

addition, I thank Dr. Ozana Fischer, Patric Baumann, Dr. Karolina

Langowska, and Adrian Nayer for fruitful discussion and suggestions

during all the time of my research at the University of Basel.

Many thank go to Gaby Persy and Vesna Olivieri for TEM

measurements and to Dr. Mariusz Grzelakowski and Dalin Wu for

providing polymers. I want to thank Dr. Dirk de Bruyn for helping me to

synthetise the peptides.

Furthermore, I want to thank all the group members for a very friendly

atmosphere in and outside the Lab. This work is based on the financial

support provided by the Swiss National Science Foundation, and the

National Centre of Competence in Nanoscale Science. This is gratefully

acknowledged. The financial support for a Short Term Scientific Mission

at the University of Antwerp is also honoured.

P. Tanner

152


153

10. Abbreviations

% Percent

°C Degree celsius

Å Angstrom

A2 Second virial coefficient

AIDS Acquired immunodeficiency syndrome

ALS Amyotrophic lateral sclerosis

AP Artificial peroxisome

AqpZ Aquaporin Z

Ar Argon

ATP Adenosine triphosphate

BPA Bis(2-pyridylmethyl)amine

CAT Catalase

CLSM Confocal laser-scanning microscopy

Co Cobalt

cpm Counts per molecule

CR Count rate

Cu Copper

Cu/ZnSOD Copper-zinc superoxide dismutase

CW Continuous wave

D Diffusion coefficient

Da Dalton

DFT Discrete Fourier transform

DIEN Diethylenetriamine

DLS Dynamic light scattering

DMSO Dimethyl sulphoxide

DNA Deoxyribonucleic acid

EEV Encapsulation efficiency based on vesicles

EGFP Enhanced green fluorescent protein

EMA European medicines agency

ENDOR Electron nuclear double resonance

EPR Electron paramagnetic resonance

EtOH Ethanol

FCS Fluorescence correlation spectroscopy

FCCS Fluorescence cross correlation spectroscopy

FDA U.S food and drug administration

Fe Iron

G Autocorrelation amplitude

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154

GSH Gluthatione

GSSG Gluthatione disulphide

HCL Hydrochloric acid

He-Ne Helium-Neon

HPLC High-performance liquid chromatography

HRP Horseradish peroxidase

HYSCORE Hyperfine sub-level correlation

IDA Iminodiacetato

K+ Potassium

K Binding constant

kb Boltzmann constant

kDa kilo Dalton

kHz kilo Hertz

KM Michaelis Menten constant

LPO Lactoperoxidase

M Weight-average molar mass

MBP-FITC Maltose binding protein- fluorescein isothiocyanate

MHz mega Hertz

min Minute

ms Millisecond

mW milli Watt

Ni Nickel

nm Nanometre

NTA Nitrilotriacetic acid

OmpF Outer membrane protein F

OPOE N-Octyl-oligo-oxyethylene

OTf Triflate

P Packing parameter

PBS Phosphate buffered saline

PDI Polydispersity

PEG Polyethylene glycol

pH Potential hydrogen

PL Laser power

PB-b-PEO Poly(butadiene)-b-poly(ethylene oxide)

PMOXA-PDMS-PMOXA poly-(2-methyloxazoline)-poly-(dimethylsiloxane)-poly-(2-

methyloxazoline)

PRX Peroxiredoxin

RFP Red fluorescent protein

Rg Radius of gyration


155

RH Hydrodynamic radius

RNA Ribonucleic acid

RNS Reactive nitrogen species

ROS Reactive oxygen species

s Second

SA Succinic anhydride

SEM Scanning electron microscopy

SLS Static light scattering

SOD Superoxide dismutase

STED Stimulated emission depletion

STORM Stochastic optical reconstruction microscopy

T Temperature

TEM Transmission electron microscopy

TFE Trifluoroethanol

TPA Tris(2-pyridylmethyl)amine

TREN Tri(2-aminoethyl)amine

TRX Thioredoxin

Vmax Maximal reaction rate

XA Xanthine

XO Xanthine oxidase

Zn Zinc

λ Wavelength

λem Emission wavelength

λex Excitation wavelength

µg micro gram

µs micro second

τd Diffusion time

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157

11. Curriculum vitae

Name Pascal Tanner

E-mail [email protected]

Telephone +41 (0) 62 534 14 99

Date of birth 22.05.1984

Nationality Switzerland

Civil status married

Hometown Werthenstein (LU)

Education

2009 – 2013 PhD in the group of Prof. Dr. C. Palivan Department of

Physical-Chemistry, University of Basel

Title: Design and development of protein-polymer

assemblies to engineer artificial organelles

2008 – 2009 Master of Science in Nanosciences in the group of

Prof. W. Meier, Department of Physical-Chemistry,

University of Basel (grade 5.9)

Title: Metal-functionalized polymer vesicles for

molecular recognition

2004 – 2007 Bachelor of Science in Nanosciences

University of Basel (grade 4.7)

1996 – 2003 Matura

Kantonsschule Olten

Work experience

06/2009 – 2013 PhD project (Physical Chemistry Department of the

University of Basel):

- Development and characterisation of

synthetic/biological nanosystems (nanoreactors and

nanocontainers)

- Writing scientific publications (articles, reviews and

book chapter)

- Presentation of results/products at international

conferences and fairs

09/2008 – 04/2009 Master`s thesis (Physical Chemistry Department of the

University of Basel):

P. Tanner

158

- Development and characterisation of nanosystems for

molecular recognition

- Short-term scientific foreign missions

01/2008 – 03/2008 Project work (Biocentre of the University of Basel):

- Structural research of subunits in F1FO ATP- synthase

- Protein cloning and expression in E. coli.

03/2004 – 09/2007 Project works (Biocentre of the University of Basel

and at PSI):

- AFM study of damaged cartilage for early diagnosis

- AFM study of effects on fracture and erosion of

Si/SiO2-Nanotowers

09/2003 – 02/2004 Employee (SBB in Olten) 100%:

- logistics

- inventory taking.

Language skills

English fluent

German first language

French basic

Social competences

My very efficient way of working, reliability and my open communication skills

allowed me to represent our group at international conferences and fairs.

Technical skills

Development and characterisation of nanosystems (nanoreactors or

nanocontainers) for antioxidant therapy or drug delivery:

- Production/Engineering of biological/synthetic formulations

- Microscopy and molecular imaging techniques (AFM, STM, optical

microscopy, CLSM, TEM)

- Spectroscopy techniques (UV/VIS, IR, EPR, NMR, FCS, FCCS, DLS, SLS)

- Protein/enzyme labelling and characterisation

- basic handling of cell cultures

- Purification techniques (dialysis, size-exclusion and fast protein liquid

chromatography)

Presentation and writing skills:


159

- Presentation of results/concepts at international conferences and fairs

- Writing scientific publications

- Broadcasting of knowledge (introduction of practicum experiments/devices to

students)

EDV:

- Microsoft Word, Excel, PowerPoint, EndNote, Origin, Corel

Leisure time activities

Successful participation at national and international MTB Cross-

Country and Marathon events

Team competitions: Stage race Craft Bike Transalp and 24h Race Davos

Oral presentations

Nanoevent NextNanoStar, Basel, Switzerland, 21.03.2013

243rd

ACS National Meeting, San Diego, CA, USA, 25.3.2012 -

29.3.2012

Swiss Soft Days 3. WORKSHOP, Department of Chemistry, University

of Fribourg, Switzerland, 20.10.2010

Poster presentations

1.) Swiss Soft Days 8. WORKSHOP, Geneve, Switzerland, 1.6.2012, Title:

Specific His6-tag attachement to metal-functionalized polymersomes relies

on molecular recognition, Pascal Tanner, Maria Ezhevskaya, Rainer

Nehring, Sabine Van Doorslaer, Wolfgang Meier, Cornelia Palivan.

2.) Hannover Messe, Hannover, Deutschland, 23.4.2012 - 27.4.2012, Title:

Multifunctional nanoscale containers for enzymes: A simple tool to detect

and defeat harmful radicals, Pascal Tanner, Cornelia G. Palivan, Wolfgang

Meier.

3.) 243rd

ACS National Meeting, San Diego, CA, USA, 25.3.2012-29.3.2012,

Title: Enzyme cascade reactions inside polymer vesicles to fight oxidative

stress, Pascal Tanner, Vimalkumar Balasubramanian, Ozana Onaca,

Wolfgang Meier, Cornelia G. Palivan.

4.) NanoBioEurope, WORKSHOP, Cork, Ireland, 21.6.2011-23.6.2011, Title:

How to fight oxidative stress with enzymes acting in tandem inside polymer

vesicles? Pascal Tanner, Vimal Balasubramanian, Ozana Onaca, Cornelia

G. Palivan.

P. Tanner

160

5.) Swiss Soft Days 4. WORKSHOP, Nestlé Research Center, Lausanne,

Switzerland, 3.2.2011, Title: Efficient antioxidant therapeutics:

nanoreactors containing enzymes/ mimics at cellular level, Pascal Tanner,

Ozana Onaca, Vimalkumar Balasubramanian, Cornelia G. Palivan.

6.) Fall Meeting of the Swiss Chemical Society 2010, ETH Zürich, Zürich,

Switzerland, 16.9.2010, Title: Metal-NTA-functionalized vesicles for

molecular recognition, Pascal Tanner, Rainer Nehring, Cornelia G.

Palivan, Wolfgang Meier.

7.) Swiss Soft Days 2. WORKSHOP, Ecole Polytechnique de Lausanne,

Lausanne, Switzerland, 23.6.2010, Title: How membrane proteins act as

gates for POLYMERC nanocontainers, Pascal Tanner, Ozana Onaca,

Patric Baumann, Cornelia G. Palivan.

8.) FUNctional Multiscale ARCHitectures Workshop, National Research

Council, Bologna, Italy, 5.5.2010 - 7.5.2010, Title: Metal-NTA-

functionalized vesicles for molecular recognition, Pascal Tanner, Rainer

Nehring, Cornelia G. Palivan, Wolfgang Meier.

9.) Fall Meeting of the Swiss Chemical Society 2009, Ecole Polytechnique

Fédérale de Lausanne, Switzerland, 4.9.2009, Title: Optimisation of Ni-

NTA-functionalized vesicles for molecular recognition in solution, Pascal

Tanner, Rainer Nehring, Cornelia G. Palivan, Wolfgang Meier.

10.) NCCR SNI Swiss Nano Basel, University of Basel, Switzerland, 11.6.2009,

Title: Nickel-NTA-functionalized vesicles for molecular recognition,

Pascal Tanner, Rainer Nehring, Cornelia G. Palivan, Wolfgang Meier.

Summer school

Summer School Essen, Germany, 22.09.2009-25.09.2009, Computational

Chemistry and Spectroscopy

Awards

- STSM grant from COST P15 action

- SCNAT/SCS Swiss Travel Award


161

Publications

1.) Tanner, P.; Balasubramanian, V.; Palivan, C. G. Aiding Nature’s

Organelles: Artificial Peroxisomes Play Their Role. Nano Letters, 2013,

DOI: 10.1021/nl401215n

2.) Itel, F.; Dinu, A.; Tanner, P.; Fischer, O.; Palivan, G. C. Nanoreactors for

biomedical applications, submitted.

3.) Heinisch, T.; Langowska, K.; Tanner, P.; Reymond, J.L. ; Palivan, C.;

Meier, W.; Ward, T. Fluoreswcence-based assay for the optimization of

artificial transfer hydrogenase activity within a biocompatible compartment

ChemCatChem., 2013, DOI: 10.1002/cctc.201200834

4.) Dobrunz, D.; Toma, A.;Tanner, P.; Pfohl, T.; Palivan C. Polymer

nanoreactors with a dual-functionality: simultaneous detoxification of

peroxynitrites and transport of oxygen Langmuir, 2012, DOI:

10.1021/la302724m

5.) Tanner, P.; Ezhevskaya, M.; Nehring, R.; Van Doorslaer, S.; Meier, W. P.;

Palivan, C. G., Specific His6-tag Attachment to Metal-Functionalized

Polymersomes Relies on Molecular Recognition. The Journal of Physical

Chemistry B 2012, 116, 10113-24.

6.) Zhang, X.; Tanner, P.; Graff, A.; Palivan, C.; Meier, W., Mimicking the

Cell Membrane with Block Copolymer Membranes. Journal of Polymer

Science, Part A: Polymer Chemistry 2012, 50, 2293-2318.

7.) Tanner, P.; Egli, S.; Balasubramanian, V.; Onaca, O.; Palivan, C. G.; Meier,

W., Can Polymeric Vesicles that Confine Enzymatic Reactions act as

Simplified Organelles? FEBS Letters 2011, 585, 1699-1706.

8.) Tanner, P.; Onaca, O.; Balasubramanian, V.; Meier, W.; Palivan, C. G.:

Enzymatic Cascade Reactions inside Polymeric Nanocontainers: A Means

to Combat Oxidative Stress. Chemistry – A European Journal 2011, 17,

4552-4560.

9.) Tanner, P.; Baumann, P.; Enea, R.; Onaca, O.; Palivan, C. G.; Meier, W.:

Polymeric Vesicles: from Drug Carriers to Nanoreactors and Artificial

Organelles Accounts of Chemical Research 2011, 44, 1039-1049.

10.) Baumann, P.; Tanner, P.; Onaca, O.; Palivan, C.G. Bio-Decorated Polymer

Membranes: A New Approach in Diagnostics and

Therapeutics. Polymers 2011, 3, 173-192.

11.) Nehring, R.; Palivan, C. G.; Moreno-Flores, S.; Mantion, A.; Tanner, P.;

Toca-Herrera, J. L.; Thuenemann, A.; Meier, W.: Protein Decorated

Membranes by Specific Molecular Interactions. Soft Matter 2010, 6, 2815-

2824.

12.) Nehring, R.; Palivan, C. G.; Casse, O.; Tanner, P.; Tuxen, J.; Meier, W.:

Amphiphilic Diblock Copolymers for Molecular Recognition: Metal-

Nitrilotriacetic Acid Functionalized Vesicles. Langmuir 2008, 25, 1122-

1130.

P. Tanner

162

References

1. Prof. Dr. Cornelia G. Palivan

Department of Chemistry, University of Basel, Switzerland

Email: [email protected]

Phone: 061 267 38 39

2. Prof. Dr. Wolfgang Meier

Department of Chemistry, University of Basel, Switzerland

Email: [email protected]

Phone: 061 267 38 02

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